Antigen-binding molecule promoting disappearance of antigens having plurality of biological activities

ABSTRACT

The present inventors newly discovered that even if an antigen-binding molecule inhibits in vitro some of the physiological activities of an antigen having two or more physiological activities without inhibiting the remaining physiological activities, the molecule can promote elimination of the antigen from blood (from serum or plasma) and as a result reduce the physiological activities in vivo, when the antigen-binding molecule is conferred with the properties: (i) of binding to human FcRn under an acidic pH range condition; (ii) of binding under a neutral pH range condition to human Fc receptor stronger than native human IgG, and (iii) that its antigen-binding activity alters according to the ion concentration.

TECHNICAL FIELD

The present invention relates to antigen-binding molecules that canpromote antigen elimination from the blood (serum or plasma), andthereby reduce the in vivo physiological activities of the antigen whichare difficult to inhibit in vitro due to multiple physiologicalactivities of the antigen, and pharmaceutical compositions comprising asan active ingredient the antigen-binding molecules.

BACKGROUND ART

There are many known examples of diseases whose onset is caused bydisruption of the balance of physiological activities maintained underhealthy conditions, due to an excessively increased plasma level of aphysiologically active substance (for example, a cytokine) relative tohealthy conditions. A possible effective means of treating such diseasesis to inhibit the physiological activity of the excessivephysiologically active substance. For example, an antibody that binds toan antigen having physiological activity, and thereby neutralizes itsphysiological activity can be an effective therapeutic agent.

However, when an antigen has two or more physiological activities,normally a single type of neutralizing antibody can inhibit only onephysiological activity. It is expected to be difficult for such anantibody to cure a disease caused by the above-described physiologicallyactive substance.

Physiologically active substances that have two or more physiologicalactivities include, for example, high mobility group box 1 (HMGB1).HMGB1 was identified as a member of the HMG family which is a nuclearprotein that contributes to the stability of higher-order DNA structureby binding to the DNA. HMGB1 consists of 215 amino acids, andstructurally it is composed of three main domains: HMG A box, HMG B box,and an acidic carboxyl-terminal domain. Normally, HMGB1 is present incells as a DNA-binding protein. However, HMGB1 is released to theoutside of inflammatory or necrotic cells through an active or passivemechanism. Released HMGB1 is known to activate a variety of cell surfacereceptors such as receptor for advanced glycation endproducts (RAGE),Toll-like receptor 4 (TLR4), and IL-1 receptor via binding to varioussubstances such as DNAs, lipopolysaccharide (LPS), and interleukin(IL)-1β, and thereby transmit the signal into cells, which leads toinduction of various inflammatory reactions (Non-patent Document 1).Furthermore, HMGB1 has been suggested to play an important role in theonset of sepsis, based on the fact that the blood level of HMGB1 waselevated in sepsis model mice administered with LPS and the mousemortality rate was decreased by administration of a polyclonal antibodyagainst HMGB1 (Non-patent Document 2). Patent Document 1 disclosespreparation of several monoclonal antibodies with high affinity forHMGB1 and that they inhibited the binding of HMGB1 to RAGE or TLR4 andreduced the mortality rate of sepsis model mice. However, there is nodescription on the acquisition of antibodies that inhibit the HMGB1activities towards both RAGE and TLR4, suggesting that it is difficultfor a single type of antibody to inhibit multiple activities of HMGB1.

Antibodies (IgGs) bind to neonatal Fc receptor (FcRn), and have longplasma retention. The binding of IgG to FcRn is observed only under anacidic condition (pH 6.0), and it is hardly observed under the neutralcondition (pH 7.4). Typically, IgG is nonspecifically incorporated intocells via endocytosis, and returns to the cell surface by binding toendosomal FcRn under the acidic condition in the endosome. Then, IgGdissociates from FcRn under the neutral condition in plasma. IgGs thatdo not bind to FcRn enter the lysosome and are degraded there. When theFcRn binding of an IgG under the acidic condition is eliminated byintroducing mutations into its Fc region, the IgG is not recycled fromthe endosome to the plasma, and as a result the plasma retention of IgGis markedly impaired. For a method of improving the plasma retention ofIgG, a method that improves the FcRn binding under acidic conditions hasbeen reported. When the FcRn binding under acidic conditions is improvedby introducing an amino acid substitution into an IgG Fc region, theefficiency of recycling from the endosome to the plasma is increased,resulting in an improvement of the plasma retention. Meanwhile, it hasbeen reported that, when the FcRn binding under the neutral condition isenhanced, IgG does not dissociate from FcRn under the neutral conditionin plasma even when it returns to the cell surface via binding to FcRnunder the acidic condition in the endosome, and consequently the plasmaretention remains unaltered or is rather worsened (Non-patent Documents3 to 5).

Recently, antibodies that bind to antigens in a pH-dependent manner havebeen reported (Patent Document 2). The antibodies, which strongly bindto antigens under the plasma neutral condition and dissociate from theantigens under the endosomal acidic condition, after being dissociatedfrom the antigen, become again capable of binding to antigens whenrecycled to the plasma via FcRn. Thus, a single antibody can bind tomultiple antigens repeatedly. Plasma retention of an antigen is muchshorter than that of an antibody which has the FcRn-mediated recyclingmechanism. Therefore, when an antigen is bound to an antibody, theplasma retention of the antigen is normally prolonged, resulting in anincrease of antigen concentration in the plasma. On the other hand, ithas been reported that the above-described antibodies which bind toantigens in a pH-dependent manner promote antigen elimination fromplasma as compared to typical antibodies because they dissociate fromthe antigens in the endosome during the FcRn-mediated recycling process(Patent Document 2). However, there is no known antibody engineeringtechnique that further improves the above-described effect of promotingantigen elimination from plasma.

PRIOR ART DOCUMENTS [Patent Documents]

-   [Patent Document 1] WO1995/002187-   [Patent Document 2] WO2009/125825

[Non-patent Documents]

-   [Non-patent Document 1] Sims G P et al., Annu Rev. Immunol. (2010)    28, 367-388.-   [Non-patent Document 2] Wang H et al., Science (1999) 285, 248-251.-   [Non-patent Document 3] Yeung Y A et al., J. Immunol. (2009) 182,    7663-71.-   [Non-patent Document 4] Datta-Mannan A et al., J. Biol. Chem. (2007)    282, 1709-17.-   [Non-patent Document 5] Dall'Acqua W F et al., J. Immunol. (2002)    169, 5171-80.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide antigen-bindingmolecules that can promote antigen elimination from the blood (serum orplasma) and thereby reduce in vivo the physiological activities of theantigen which are difficult to inhibit with a single type ofantigen-binding molecule in vitro because the antigen has two or morephysiological activities. Another objective of the present invention isto provide methods for producing the antigen-binding molecules andpharmaceutical compositions comprising the antigen-binding molecules asan active ingredient.

Means for Solving the Problems

The present inventors conducted dedicated studies. As a result, thepresent inventors newly revealed that an antigen-binding molecule thatinhibits some of the physiological activities of antigens with two ormore physiological activities but does not inhibit the remainingphysiological activities in vitro can promote antigen elimination fromblood (serum or plasma), and reduce the physiological activities invivo, by conferring the antigen-binding molecule with the properties of:

-   -   (i) binding to human neonatal Fc receptor (FcRn) under an acidic        pH range condition;    -   (ii) binding to human FcRn and/or human Fcγ receptor more        strongly than native human IgG under a neutral pH range        condition; and    -   (iii) altering the antigen-binding activity according to the ion        concentration condition.

The present invention is based on the above findings, and specificallyrelates to:

[1] an antigen-binding molecule that reduces plasma antigenconcentration, wherein the antigen-binding molecule has characteristics(1) to (6) below:

-   -   (1) the antigen-binding molecule comprises an antigen-binding        domain and at least one receptor-binding domain;    -   (2) under an acidic pH range condition, the receptor-binding        domain has human neonatal Fc Receptor (FcRn)-binding activity;    -   (3) under a neutral pH range condition, the human Fc        receptor-binding activity of the receptor-binding domain is        higher than human Fc receptor-binding activity of native human        IgG;    -   (4) an antigen-binding activity of the antigen-binding domain is        altered according to the ion concentration condition;    -   (5) the antigen has two or more physiological activities; and    -   (6) one or more physiological activities of the antigen are        inhibited by binding of the antigen-binding molecule, while at        least one type of physiological activity of the antigen is        maintained;        [2] the antigen-binding molecule of [1], characterized in that        it inhibits the activity of an antigen to bind one or more types        of target molecules by binding to the antigen while allowing the        antigen to maintain the binding activity to at least one type of        target molecule;        [3] the antigen-binding molecule of [1] or [2], wherein the        reduction of plasma antigen concentration is due to the        promotion of antigen uptake into cells;        [4] the antigen-binding molecule of any one of [1] to [3],        characterized in that the decrease of antigen concentration in        plasma results in reduction of the physiological activity of the        antigen in vivo;        [5] the antigen-binding molecule of any one of [1] to [4],        wherein the antigen is high mobility group box 1 (HMGB1);        [6] the antigen-binding molecule of [5], which inhibits the        binding between HMGB1 and receptor for advanced glycation        endproducts (RAGE);        [7] the antigen-binding molecule of [5] or [6], which inhibits        the binding between HMGB1 and Toll-like receptor 4 (TLR4);        [8] the antigen-binding molecule of any one of [1] to [4],        wherein the antigen is connective tissue growth factor (CTGF);        [9] the antigen-binding molecule of any one of [1] to [8],        wherein the human Fc receptor is human FcRn;        [10] the antigen-binding molecule of [9], wherein the        receptor-binding domain comprises an Fc region in which at least        one amino acid in the IgG Fc region is altered;        [11] the antigen-binding molecule of [10], wherein the amino        acid alteration in the IgG Fc region is an alteration of at        least one amino acid selected from positions:        234, 235, 236, 237, 238, 239, 244, 245, 248, 249, 250, 251, 252,        253, 254, 255, 256, 257, 258, 260, 262, 265, 267, 270, 272, 274,        279, 280, 282, 283, 284, 285, 286, 288, 289, 293, 295, 297, 298,        303, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318,        325, 326, 327, 328, 329, 330, 332, 334, 338, 339, 340, 341, 343,        345, 360, 361, 362, 375, 376, 377, 378, 380, 382, 384, 385, 386,        387, 389, 390, 391, 413, 422, 423, 424, 427, 428, 430, 431, 433,        434, 435, 436, 437, 438, 440, and 442 (EU numbering);        [12] the antigen-binding molecule of [11], wherein the amino        acid alteration in the IgG Fc region is at least one amino acid        alteration selected from the alterations: wherein according to        EU numbering,        the amino acid at position 234 is Arg;        the amino acid at position 235 is Gly, Lys, or Arg;        the amino acid at position 236 is Ala, Asp, Lys, or Arg;        the amino acid at position 237 is Lys, Met, or Arg;        the amino acid at position 238 is Ala, Asp, Lys, Leu, or Arg;        the amino acid at position 239 is Asp or Lys;        the amino acid at position 244 is Leu;        the amino acid at position 245 is Arg;        the amino acid at position 248 is Ile or Tyr;        the amino acid at position 249 is Pro;        the amino acid at position 250 is Ala, Glu, Phe, Ile, Met, Gln,        Ser, Val, Trp, Gly, His, Leu, Asn, or Tyr;        the amino acid at position 251 is Arg, Asp, Glu, or Leu;        the amino acid at position 252 is Phe, Ser, Thr, Trp, or Tyr;        the amino acid at position 253 is Val;        the amino acid at position 254 is Ala, Gly, His, Ile, Gln, Ser,        Val, or Thr;        the amino acid at position 255 is Ala, Asp, Phe, His, Ile, Lys,        Leu, Met, Asn, Gln, Arg, Gly, Ser, Trp, Tyr, or Glu;        the amino acid at position 256 is Ala, Asp, Glu, Arg, Asn, Pro,        Thr, Ser, or Gln;        the amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn,        Ser, Thr, or Val;        the amino acid at position 258 is Asp or His;        the amino acid at position 260 is Ser;        the amino acid at position 262 is Leu;        the amino acid at position 265 is Ala;        the amino acid at position 267 is Met or Leu;        the amino acid at position 270 is Lys or Phe;        the amino acid at position 272 is Ala, Leu, or Arg;        the amino acid at position 274 is Ala;        the amino acid at position 279 is Leu, Ala, Asp, Gly, His, Met,        Asn, Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 280 is Ala, Gly, His, Lys, Asn, Gln,        Arg, Ser, Thr, or Glu;        the amino acid at position 282 is Ala or Asp;        the amino acid at position 283 is Ala, Asp, Phe, Gly, His, Ile,        Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 284 is Lys;        the amino acid at position 285 is Asn;        the amino acid at position 286 is Ala, Asp, Phe, Gly, His, Ile,        Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or Glu;        the amino acid at position 288 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Leu, Met, Asn, Pro, Gln, Arg, Val, Trp, Tyr, or Ser;        the amino acid at position 289 is His;        the amino acid at position 293 is Val;        the amino acid at position 295 is Met;        the amino acid at position 297 is Ala;        the amino acid at position 298 is Gly;        the amino acid at position 303 is Ala;        the amino acid at position 305 is Ala or Thr;        the amino acid at position 307 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;        the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro,        Gln, or Thr;        the amino acid at position 309 is Ala, Asp, Glu, Pro, His, or        Arg;        the amino acid at position 311 is Ala, His, Glu, Lys, Leu, Met,        Ser, Val, Trp, or Ile;        the amino acid at position 312 is Ala, Asp, Pro, or His;        the amino acid at position 313 is Tyr or Phe;        the amino acid at position 314 is Ala, Leu, Lys, or Arg;        the amino acid at position 315 is Ala, Asp, Glu, Phe, Gly, Ile,        Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or His;        the amino acid at position 316 is Ala, Glu, Phe, His, Ile, Lys,        Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Asp;        the amino acid at position 317 is Ala or Pro;        the amino acid at position 318 is Asn or Thr;        the amino acid at position 325 is Ala, Gly, Met, Leu, Ile, or        Ser;        the amino acid at position 326 is Asp;        the amino acid at position 327 is Gly;        the amino acid at position 328 is Arg, Asp, Glu, or Tyr;        the amino acid at position 329 is Lys or Arg;        the amino acid at position 330 is Leu;        the amino acid at position 332 is Glu, Phe, His, Lys, Leu, Met,        Arg, Ser, Trp, or Val;        the amino acid at position 334 is Leu;        the amino acid at position 338 is Ala;        the amino acid at position 339 is Asn, Thr, or Trp;        the amino acid at position 340 is Ala;        the amino acid at position 341 is Pro;        the amino acid at position 343 is Glu, His, Lys, Gln, Arg, Thr,        or Tyr;        the amino acid at position 345 is Ala;        the amino acid at position 360 is His;        the amino acid at position 361 is Ala;        the amino acid at position 362 is Ala;        the amino acid at position 375 is Ala or Arg;        the amino acid at position 376 is Ala, Gly, Ile, Met, Pro, Thr,        or Val;        the amino acid at position 377 is Lys;        the amino acid at position 378 is Asp, Asn, or Val;        the amino acid at position 380 is Ala, Asn, Thr, or Ser;        the amino acid at position 382 is Ala, Phe, His, Ile, Lys, Leu,        Met, Asn, Gln, Arg, Ser, Thr, Trp, Tyr, or Val;        the amino acid at position 384 is Ala;        the amino acid at position 385 is Ala, Gly, Lys, Ser, Thr, Asp,        His, or Arg;        the amino acid at position 386 is Arg, Asp, Ile, Met, Ser, Thr,        Lys, or Pro;        the amino acid at position 387 is Ala, Arg, His, Pro, Ser, Thr,        or Glu;        the amino acid at position 389 is Ala, Asn, Pro, or Ser;        the amino acid at position 390 is Ala;        the amino acid at position 391 is Ala;        the amino acid at position 413 is Ala;        the amino acid at position 423 is Asn;        the amino acid at position 424 is Ala or Glu;        the amino acid at position 427 is Asn;        the amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys,        Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr;        the amino acid at position 431 is His or Asn;        the amino acid at position 433 is Arg, Gln, His, Ile, Pro, Ser,        or Lys;        the amino acid at position 434 is Ala, Phe, Gly, Met, His, Ser,        Trp, or Tyr;        the amino acid at position 435 is Lys, Arg, or Asn;        the amino acid at position 436 is Ala, His, Ile, Leu, Glu, Phe,        Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp, or Val;        the amino acid at position 437 is Arg;        the amino acid at position 438 is Lys, Leu, Thr, or Trp;        the amino acid at position 440 is Lys; and        the amino acid at position 442 is Lys;        [13] the antigen-binding molecule of any one of [10] to [12],        wherein the IgG Fc region is the Fc region of a nonhuman        animal-derived IgG;        [14] the antigen-binding molecule of any one of [10] to [12],        wherein the IgG Fc region is the Fc region of a human-derived        IgG;        [15] the antigen-binding molecule of any one of [1] to [8],        wherein the human Fc receptor is a human Fcγ receptor;        [16] the antigen-binding molecule of [15], wherein the        receptor-binding domain comprises an Fc region in which at least        one amino acid in the IgG Fc region is altered;        [17] the antigen-binding molecule of [16], wherein the amino        acid alteration in the IgG Fc region is an alteration of at        least one amino acid selected from positions:        221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235,        236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,        251, 252, 254, 255, 256, 257, 258, 260, 262, 263, 264, 265, 266,        267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280,        281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295,        296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309,        311, 312, 313, 314, 315, 316, 317, 318, 320, 322, 323, 324, 325,        326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339,        341, 343, 375, 376, 377, 378, 379, 380, 382, 385, 386, 387, 389,        392, 396, 421, 423, 427, 428, 429, 430, 431, 433, 434, 436, 438,        440; and 442 (EU numbering);        [18] the antigen-binding molecule of [17], wherein the amino        acid alteration in the IgG Fc region is at least one amino acid        alteration selected from the alterations: wherein, according to        EU numbering,        the amino acid at position 221 is Lys or Tyr;        the amino acid at position 222 is Phe, Trp, Glu, or Tyr;        the amino acid at position 223 is Phe, Trp, Glu, or Lys;        the amino acid at position 224 is Phe, Trp, Glu, or Tyr;        the amino acid at position 225 is Glu, Lys, or Trp;        the amino acid at position 227 is Glu, Gly, Lys, or Tyr;        the amino acid at position 228 is Glu, Gly, Lys, or Tyr;        the amino acid at position 230 is Ala, Glu, Gly, or Tyr;        the amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr;        the amino acid at position 232 is Glu, Gly, Lys, or Tyr;        the amino acid at position 233 is Ala, Asp, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 234 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 235 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 236 is Ala, Asp, Glu, Phe, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 237 is Ala, Asp, Glu, Phe, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 238 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 239 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;        the amino acid at position 240 is Ala, Ile, Met, or Thr;        the amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or        Tyr;        the amino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp,        or Tyr;        the amino acid at position 244 is His;        the amino acid at position 245 is Ala;        the amino acid at position 246 is Asp, Glu, His, or Tyr,        the amino acid at position 247 is Ala, Phe, Gly, His, Ile, Leu,        Met, Thr, Val, or Tyr;        the amino acid at position 249 is Glu, His, Gln, or Tyr;        the amino acid at position 250 is Glu or Gln;        the amino acid at position 251 is Phe;        the amino acid at position 254 is Phe, Met, or Tyr;        the amino acid at position 255 is Glu, Leu, or Tyr;        the amino acid at position 256 is Ala, Met, or Pro;        the amino acid at position 258 is Asp, Glu, His, Ser, or Tyr;        the amino acid at position 260 is Asp, Glu, His, or Tyr;        the amino acid at position 262 is Ala, Glu, Phe, Ile, or Thr;        the amino acid at position 263 is Ala, Ile, Met, or Thr;        the amino acid at position 264 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 265 is Ala, Glu, Leu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or        Tyr;        the amino acid at position 266 is Ala, Phe, Ile, Leu, Met, or        Thr;        the amino acid at position 267 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;        the amino acid at position 268 is Ala, Asp, Glu, Phe, Gly, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Trp;        the amino acid at position 269 is Asp, Phe, Gly, His, Ile, Lys,        Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 270 is Glu, Phe, Gly, His, Ile, Leu,        Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 271 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 272 is Asp, Phe, Gly, His, Ile, Lys,        Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 273 is Phe or Ile;        the amino acid at position 274 is Asp, Glu, Phe, Gly, His, Ile,        Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 275 is Leu or Trp;        the amino acid at position 276 is Asp, Glu, Phe, Gly, His, Ile,        Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 278 is Asp, Glu, Gly, His, Ile, Lys,        Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;        the amino acid at position 279 is Ala;        the amino acid at position 280 is Ala, Gly, His, Lys, Leu, Pro,        Gln, Trp, or Tyr;        the amino acid at position 281 is Asp, Lys, Pro, or Tyr;        the amino acid at position 282 is Glu, Gly, Lys, Pro, or Tyr;        the amino acid at position 283 is Ala, Gly, His, Ile, Lys, Leu,        Met, Pro, Arg, or Tyr;        the amino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or        Tyr;        the amino acid at position 285 is Asp, Glu, Lys, Gln, Trp, or        Tyr;        the amino acid at position 286 is Glu, Gly, Pro, or Tyr;        the amino acid at position 288 is Asn, Asp, Glu, or Tyr;        the amino acid at position 290 is Asp, Gly, His, Leu, Asn, Ser,        Thr, Trp, or Tyr;        the amino acid at position 291 is Asp, Glu, Gly, His, Ile, Gln,        or Thr;        the amino acid at position 292 is Ala, Asp, Glu, Pro, Thr, or        Tyr;        the amino acid at position 293 is Phe, Gly, His, Ile, Leu, Met,        Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 294 is Phe, Gly, His, Ile, Lys, Leu,        Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 296 is Ala, Asp, Glu, Gly, His, Ile,        Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val;        the amino acid at position 297 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 298 is Ala, Asp, Glu, Phe, His, Ile,        Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr;        the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;        the amino acid at position 300 is Ala, Asp, Glu, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;        the amino acid at position 301 is Asp, Glu, His, or Tyr;        the amino acid at position 302 is Ile;        the amino acid at position 303 is Asp, Gly, or Tyr;        the amino acid at position 304 is Asp, His, Leu, Asn, or Thr;        the amino acid at position 305 is Glu, Ile, Thr, or Tyr;        the amino acid at position 311 is Ala, Asp, Asn, Thr, Val or        Tyr;        the amino acid at position 313 is Phe;        the amino acid at position 315 is Leu;        the amino acid at position 317 is Glu or Gln;        the amino acid at position 318 is His, Leu, Asn, Pro, Gln, Arg,        Thr, Val, or Tyr;        the amino acid at position 320 is Asp, Phe, Gly, His, Ile, Leu,        Asn, Pro, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 322 is Ala, Asp, Phe, Gly, His, Ile,        Pro, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 323 is Ile, Leu, or Met;        the amino acid at position 324 is Asp, Phe, Gly, His, Ile, Leu,        Met, Pro, Arg, Thr, Val, Trp, or Tyr;        the amino acid at position 325 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 326 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 327 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr;        the amino acid at position 328 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 330 is Cys, Glu, Phe, Gly, His, Ile,        Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 331 is Asp, Phe, His, Ile, Leu, Met,        Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 332 is Ala, Asp, Glu, Phe, Gly, His,        Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 333 is Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, or Tyr;        the amino acid at position 334 is Ala, Glu, Phe, His, Ile, Leu,        Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 335 is Asp, Phe, Gly, His, Ile, Leu,        Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr;        the amino acid at position 336 is Glu, Lys, or Tyr;        the amino acid at position 337 is Asp, Glu, His, or Asn;        the amino acid at position 339 is Asp, Phe, Gly, Ile, Lys, Met,        Asn, Gln, Arg, Ser, or Thr;        the amino acid at position 376 is Ala or Val;        the amino acid at position 377 is Gly or Lys;        the amino acid at position 378 is Asp;        the amino acid at position 379 is Asn;        the amino acid at position 380 is Ala, Asn, or Ser;        the amino acid at position 382 is Ala or Ile;        the amino acid at position 385 is Glu;        the amino acid at position 392 is Thr;        the amino acid at position 396 is Asp, Glu, Phe, Ile, Lys, Leu,        Met, Gln, Arg, or Tyr;        the amino acid at position 421 is Lys;        the amino acid at position 427 is Asn;        the amino acid at position 428 is Phe or Leu;        the amino acid at position 429 is Met;        the amino acid at position 434 is Trp;        the amino acid at position 436 is Ile; and        the amino acid at position 440 is Gly, His, Ile, Leu, or Tyr;        [19] the antigen-binding molecule of any one of [16] to [18],        wherein the human Fcγ receptor is FcγRIa, FcγRIIa, FcγRIIb, or        FcγRIIIa;        [20] the antigen-binding molecule of [17], wherein the amino        acid alteration in the IgG Fc region is an alteration wherein        the amino acid at position 238 is Asp and the amino acid at        position 271 is Gly according to EU numbering;        [21] the antigen-binding molecule of [20], wherein the IgG Fc        region comprises an additional alteration of at least one amino        acid selected from positions:        233, 234, 237, 244, 245, 249, 250, 251, 252, 254, 255, 256, 257,        258, 260, 262, 264, 265, 266, 267, 268, 269, 270, 272, 279, 283,        285, 286, 288, 293, 296, 307, 308, 309, 311, 312, 314, 316, 317,        318, 326, 327, 330, 331, 332, 333, 339, 341, 343, 375, 376, 377,        378, 380, 382, 385, 386, 387, 389, 396, 423, 427, 428, 430, 431,        433, 434, 436, 438, 440, and 442 according to EU numbering;        [22] the antigen-binding molecule of [21], wherein the amino        acid alteration in the IgG Fc region is at least one amino acid        alteration selected from the alterations: wherein according to        EU numbering,        the amino acid at position 233 is Asp;        the amino acid at position 234 is Tyr;        the amino acid at position 237 is Asp;        the amino acid at position 264 is Ile;        the amino acid at position 265 is Glu;        the amino acid at position 266 is Phe, Met, or Leu;        the amino acid at position 267 is Ala, Glu, Gly, or Gln;        the amino acid at position 268 is Asp or Glu;        the amino acid at position 269 is Asp;        the amino acid at position 272 is Asp, Phe, Ile, Met, Asn, or        Gln;        the amino acid at position 296 is Asp;        the amino acid at position 326 is Ala or Asp;        the amino acid at position 327 is Gly;        the amino acid at position 330 is Lys or Arg;        the amino acid at position 331 is Ser;        the amino acid at position 332 is Thr,        the amino acid at position 333 is Thr, Lys, or Arg; and        the amino acid at position 396 is Asp, Glu, Phe, Ile, Lys, Leu,        Met, Gln, Arg, or Tyr;        [23] the antigen-binding molecule of any one of [20] to [22],        wherein the IgG Fc region comprises an additional alteration of        at least one amino acid selected from positions:        244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262,        270, 272, 279, 283, 285, 286, 288, 293, 307, 308, 309, 311, 312,        314, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380,        382, 385, 386, 387, 389, 423, 427, 428, 430, 431, 433, 434, 436,        438, 440, and 442 (EU numbering);        [24] the antigen-binding molecule of [23], wherein the amino        acid alteration in the IgG Fc region is at least one amino acid        alteration selected from the alterations: wherein according to        EU numbering,        the amino acid at position 244 is Leu;        the amino acid at position 245 is Arg;        the amino acid at position 249 is Pro;        the amino acid at position 250 is Gln or Glu;        the amino acid at position 251 is Arg, Asp, Glu, or Leu;        the amino acid at position 252 is Phe, Ser, Thr, or Tyr;        the amino acid at position 254 is Ser or Thr;        the amino acid at position 255 is Arg, Gly, Ile, or Leu;        the amino acid at position 256 is Ala, Arg, Asn, Asp, Gln, Glu,        Pro, or Thr;        the amino acid at position 257 is Ala, Ile, Met, Asn, Ser, or        Val;        the amino acid at position 258 is Asp;        the amino acid at position 260 is Ser;        the amino acid at position 262 is Leu;        the amino acid at position 270 is Lys;        the amino acid at position 272 is Leu or Arg;        the amino acid at position 279 is Ala, Asp, Gly, His, Met, Asn,        Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 283 is Ala, Asp, Phe, Gly, His, Ile,        Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;        the amino acid at position 285 is Asn;        the amino acid at position 286 is Phe;        the amino acid at position 288 is Asn or Pro;        the amino acid at position 293 is Val;        the amino acid at position 307 is Ala, Glu, Gln, or Met;        the amino acid at position 308 is Ile, Pro, or Thr;        the amino acid at position 309 is Pro,        the amino acid at position 311 is Ala, Glu, Ile, Lys, Leu, Met,        Ser, Val, or Trp;        the amino acid at position 312 is Ala, Asp, or Pro;        the amino acid at position 314 is Ala or Leu;        the amino acid at position 316 is Lys;        the amino acid at position 317 is Pro;        the amino acid at position 318 is Asn or Thr;        the amino acid at position 332 is Phe, His, Lys, Leu, Met, Arg,        Ser, or Trp;        the amino acid at position 339 is Asn, Thr, or Trp;        the amino acid at position 341 is Pro;        the amino acid at position 343 is Glu, His, Lys, Gln, Arg, Thr,        or Tyr;        the amino acid at position 375 is Arg;        the amino acid at position 376 is Gly, Ile, Met, Pro, Thr, or        Val;        the amino acid at position 377 is Lys;        the amino acid at position 378 is Asp, Asn, or Val;        the amino acid at position 380 is Ala, Asn, Ser, or Thr;        the amino acid at position 382 is Phe, His, Ile, Lys, Leu, Met,        Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;        the amino acid at position 385 is Ala, Arg, Asp, Gly, His, Lys,        Ser, or Thr;        the amino acid at position 386 is Arg, Asp, Ile, Lys, Met, Pro,        Ser, or Thr;        the amino acid at position 387 is Ala, Arg, His, Pro, Ser, or        Thr;        the amino acid at position 389 is Asn, Pro, or Ser;        the amino acid at position 423 is Asn;        the amino acid at position 427 is Asn;        the amino acid at position 428 is Leu, Met, Phe, Ser, or Thr;        the amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys,        Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr;        the amino acid at position 431 is His or Asn;        the amino acid at position 433 is Arg, Gln, His, Ile, Lys, Pro,        or Ser;        the amino acid at position 434 is Ala, Gly, His, Phe, Ser, Trp,        or Tyr;        the amino acid at position 436 is Arg, Asn, His, Ile, Leu, Lys,        Met, or Thr;        the amino acid at position 438 is Lys, Leu, Thr, or Trp;        the amino acid at position 440 is Lys; and        the amino acid at position 442 is Lys;        [25] the antigen-binding molecule of any one of [16] to [24],        wherein the IgG Fc region is an Fc region of nonhuman        animal-derived IgG;        [26] the antigen-binding molecule of any one of [16] to [24],        wherein the IgG Fc region is an Fc region of human-derived IgG;        [27] the antigen-binding molecule of any one of [1] to [26],        wherein the ion concentration is a hydrogen-ion concentration        (pH) and the antigen-binding activity is lower under an acidic        pH range condition than under a neutral pH range condition;        [28] the antigen-binding molecule of [27], wherein the acidic pH        is endosomal pH;        [29] the antigen-binding molecule of [27] or [28], wherein the        neutral pH is plasma pH;        [30] the antigen-binding molecule of any one of [27] to [29],        wherein the acidic pH is pH 5.5 to 6.5 and the neutral pH is pH        7.0 to 8.0;        [31] the antigen-binding molecule of any one of [27] to [30],        wherein the ratio of antigen-binding activity under an acidic pH        range condition and a neutral pH range condition is 2 or more in        the value of KD (acidic pH)/KD (neutral pH);        [32] the antigen-binding molecule of any one of [27] to [31],        wherein at least one amino acid is substituted with histidine        and/or at least one histidine is inserted in the antigen-binding        domain;        [33] the antigen-binding molecule of any one of [27] to [32],        wherein additionally the antigen-binding activity is lower under        a low calcium ion concentration condition than under a high        calcium ion concentration condition;        [34] the antigen-binding molecule of any one of [1] to [26],        wherein the ion concentration is a calcium ion concentration and        the antigen-binding activity is lower under a low calcium ion        concentration condition than under a high calcium ion        concentration condition;        [35] the antigen-binding molecule of [33] or [34], wherein the        low calcium ion concentration is a calcium ion concentration in        the endosome;        [36] the antigen-binding molecule of any one of [33] to [35],        wherein the high calcium ion concentration is a calcium ion        concentration in the plasma;        [37] the antigen-binding molecule of any one of [33] to [36],        wherein the low calcium ion concentration is calcium ion        concentration of 0.1 μM to 30 μM, and the high calcium ion        concentration is calcium ion concentration of 100 μM to 10 mM;        [38] the antigen-binding molecule of any one of [33] to [37],        wherein the ratio of the antigen-binding activity under a low        calcium ion concentration condition and under a high calcium ion        concentration is 2 or more in the value of KD (low calcium ion        concentration)/KD (high calcium ion concentration);        [39] the antigen-binding molecule of any one of [1] to [38],        wherein the antigen-binding domain is obtained from a library;        [40] the antigen-binding molecule of any one of [1] to [39],        wherein the antigen-binding molecule is an antibody;        [41] the antigen-binding molecule of [40], wherein the antibody        is any one of a chimeric antibody, a humanized antibody, and a        human antibody;        [42] a pharmaceutical composition comprising as an active        ingredient the antigen-binding molecule of any one of [1] to        [41];        [43] the pharmaceutical composition of [42] for treating a        disease for which one of the causes is assumed to be the        antigen;        [44] the pharmaceutical composition of [43], wherein the antigen        is HMGB1;        [45] the pharmaceutical composition of [43] or [44], wherein the        disease is sepsis;        [46] the pharmaceutical composition of [43], wherein the antigen        is CTGF;        [47] the pharmaceutical composition of [43] or [46], wherein the        disease is fibrosis;        [48] a method for producing the antigen-binding molecule of [1],        which comprises the steps of:    -   (a) selecting an antigen having two or more physiological        activities;    -   (b) obtaining an antigen-binding domain;    -   (c) obtaining at least one receptor-binding domain;    -   (d) selecting a domain of which antigen-binding activity changes        according to the ion concentration condition from the        antigen-binding domain obtained in step (b);    -   (e) selecting from the receptor-binding domain obtained in step        (c), a domain that has human FcRn-binding activity under an        acidic pH range condition and of which binding activity to human        Fc receptor under a neutral pH range condition is higher than        the human Fc receptor-binding activity of native human IgG;    -   (f) preparing an antigen-binding molecule in which the        antigen-binding domain selected in step (d) is linked to the        receptor-binding domain selected in step (e); and    -   (g) selecting from the antigen-binding molecule prepared in step        (f), an antigen-binding molecule that inhibits one or more        physiological activities of the antigen by binding to the        antigen while allowing the antigen to maintain at least one type        of physiological activity;        [49] the method of [48], wherein step (b) is the step of        isolating a domain that binds to the antigen and then altering        at least one amino acid in the domain;        [50] the method of [48] or [49], wherein step (c) is the step of        isolating a domain that binds to human FcRn and then altering at        least one amino acid in the domain;        [51] the method of any one of [48] to [50], wherein step (f) is        the step of: (f) constructing a polynucleotide wherein a        polynucleotide encoding the antigen-binding domain selected in        step (d) is linked to a polynucleotide encoding the        receptor-binding domain selected in step (e), and producing an        antigen-binding molecule wherein the antigen-binding domain        selected in step (d) is linked to the receptor-binding domain        selected in step (e) by using the constructed polynucleotide;        [52] the method of any one of [48] to [51], wherein step (g) is        the step of:        (g) selecting from the antigen-binding molecule prepared step        (f), an antigen-binding molecule that inhibits one or more        binding activities of the antigen to target molecules by binding        to the antigen while allowing the antigen to maintain at least        one type of binding activity to target molecules; and        [53] the method of any one of [48] to [52], wherein the human Fc        receptor is human FcRn or human Fcγ receptor.

The present invention also relates to:

[54] a method for decreasing a plasma antigen concentration byadministering the antigen-binding molecule of any one of [1] to [41], oran antigen-binding molecule produced by the production method of any oneof [48] to [53];[55] a method for promoting an antigen uptake into a cell byadministering the antigen-binding molecule of any one of [1] to [41], oran antigen-binding molecule produced by the production method of any oneof [48] to [53];[56] a method for reducing the physiological activity of an antigen invivo by administering the antigen-binding molecule of any one of [1] to[41], or an antigen-binding molecule produced by the production methodof any one of [48] to [53];[57] the method of any one of [54] to [56], wherein the antigen-bindingmolecule is an antibody;[58] the method of [57], wherein the antibody is a chimeric antibody,humanized antibody, or human antibody;[59] an agent for treating a disease, which comprises as an activeingredient the antigen-binding molecule of any one of [1] to [41], or anantigen-binding molecule produced by the production method of any one of[48] to [53];[60] the therapeutic agent of [59], wherein the antigen is HMGB1;[61] the therapeutic agent of [59] or [60], wherein the disease issepsis;[62] the therapeutic agent of [59], wherein the antigen is CTGF;[63] the therapeutic agent of [59] or [62], wherein the disease isfibrosis; and[64] a kit for use in the method of any one of [54] to [58], whichcomprises the antigen-binding molecule of any one of [1] to [41], or anantigen-binding molecule produced by the production method of any one of[48] to [53].

The present invention further relates to:

[101] a method of screening for an antibody whose antigen-bindingactivity alters depending on condition, which comprises the steps of:

-   -   (a) preparing an antibody-producing cell;    -   (b) contacting the cell of (a) with an antigen under the first        condition;    -   (c) selecting from the cell of step (b) a cell to which a        predetermined amount of antigen or more is bound;    -   (d) exposing the cell of step (c) to the second condition; and    -   (e) selecting from the cell of step (d) a cell to which the        binding amount of antigen is decreased as compared to step (c);        [102] the method of [101], which comprises the steps of:    -   (a) preparing an antibody-producing cell;    -   (b) contacting an antigen with the cell of (a) under the first        condition;    -   (c) contacting an anti-IgG antibody with the cell of step (b);    -   (d) selecting from the cell of step (c) a cell to which a        predetermined amount of antigen or more is bound and a        predetermined amount of anti-IgG antibody or more is bound;    -   (e) exposing the cell of step (d) to the second condition; and    -   (f) selecting from the cell of step (e) a cell to which the        binding amount of antigen is decreased as compared to step (d);        [103] the method of [102], which comprises the steps of:    -   (a) preparing an antibody-producing cell;    -   (b) contacting an antigen with the cell of (a) under the first        condition;    -   (c) enriching a cell that is bound to an antigen from the cell        of step (b);    -   (d) contacting an anti-IgG antibody with the cell of step (c);    -   (e) selecting from the cell of step (d) a cell to which a        predetermined amount of antigen or more is bound and a        predetermined amount of anti-IgG antibody or more is bound;    -   (f) exposing the cell of step (e) to the second condition; and    -   (g) selecting from the cell of step (f) a cell to which the        binding amount of antigen is decreased as compared to step (e);        [104] the method of any one of [101] to [103], wherein the first        and second conditions are ion concentration conditions;        [105] the method of [104], wherein the first condition is a        neutral pH and the second condition is an acidic pH;        [106] the method of [104], wherein the first condition is a high        calcium ion concentration and the second condition is a low        calcium ion concentration;        [107] the method of [104], wherein the first condition is a        neutral pH and a high calcium ion concentration, and the second        condition is an acidic pH and a low calcium ion concentration;        [108] the method of [105] or [107], wherein the neutral pH is pH        7.0 to 8.0 and the acidic pH is pH 5.5 to 6.5;        [109] the method of any one of [106] to [108], wherein the high        calcium ion concentration is a calcium ion concentration of 100        μM to 10 mM, and the low calcium ion concentration is the same        as or lower than the high calcium ion concentration;        [110] the method of any one of [101] to [109], wherein the        antibody-producing cell is a cell collected from blood, spleen,        and/or lymph node;        [111] the method of any one of [101] to [110], wherein the        antibody-producing cell is a B cell;        [112] the method of [111], wherein the antibody-producing cell        is a rabbit B cell;        [113] the method of any one of [101] to [112], wherein the        antigen and/or anti-IgG antibody is fluorescently labeled;        [114] the method of any one of [101] to [113], wherein the step        of selecting a cell is carried out by using FACS; and        [115] the method of any one of [101] to [114], wherein the step        of enriching a cell is carried out by using MACS.

The present invention also relates to:

[a] methods for treating diseases for which one of the causes might bean antigen that has physiological activities, methods for reducingplasma antigen concentration, methods for promoting antigen uptake intocells, or methods for reducing the physiological activities of anantigen in vivo, which comprise the step of administering anantigen-binding molecule of the present invention;[b] therapeutic agents for diseases for which one of the causes might bean antigen that has physiological activities, agents for reducing plasmaantigen concentration, agents for promoting antigen uptake into cells,or agents for reducing the physiological activities of an antigen invivo, which comprise an antigen-binding molecule of the presentinvention as an active ingredient;[c] antigen-binding molecules of the present invention for use inmethods for treating diseases for which one of the causes might be anantigen that has physiological activities, methods for reducing plasmaantigen concentration, methods for promoting antigen uptake into cells,or methods for reducing the physiological activities of an antigen invivo;[d] use of an antigen-binding molecule of the present invention inproducing therapeutic agents for diseases for which one of the causesmight be an antigen that has physiological activities, agents forreducing plasma antigen concentration, agents for promoting antigenuptake into cells, or agents for reducing the physiological activitiesof an antigen in vivo; and[e] processes for producing therapeutic agents for diseases for whichone of the causes might be an antigen that has physiological activities,agents for reducing plasma antigen concentration, agents for promotingantigen uptake into cells, or agents for reducing the physiologicalactivities of an antigen in vivo, which comprise the step of using anantigen-binding molecule of the present invention.

Examples of diseases include diseases for which one of the causes isHMGB1, CTGF, or IgE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of FACS sorting for B cells that produceanti-HMGB1 antibodies. (A) shows a result of the first sorting under aneutral pH and high calcium ion concentration condition. (B) shows aresult of the second sorting under an acidic pH and low calcium ionconcentration condition. Numbers (1, 2, and 3) in the diagram indicategate numbers.

FIG. 2 presents plotted graphs of the HMGB1-binding activity ofanti-HMGB1 antibodies. (A), (B), and (C) shows a result of plottingantibodies produced by B cells derived from Gate 1, Gate 2, and Gate 3in FACS sorting, respectively.

FIG. 3 is a graph showing the number of antibodies whose antigen-bindingability does not depend on pH and/or calcium ion concentration (grayarea), and the number of antibodies whose antigen-binding abilitydepends on pH and/or calcium ion concentration (black area) amongantibodies produced by B cells derived from Gate 1, Gate 2, and Gate 3.

FIG. 4-1 is a diagram showing Biacore sensorgrams for anti-HMGB1antibodies (HMG233-IgG1 and HMG236-IgG1) and human HMGB1.

FIG. 4-2 is a continuation diagram of FIG. 4-1. The diagram showssensorgrams for anti-HMGB1 antibodies (HMG481-IgG1 and HMG487-IgG1) andhuman HMG1.

FIG. 5 is a graph showing a result of RAGE ELISA for anti-HMGB1antibodies (HMG233, HMG236, HMG481, HMG487, and HMG446). The absorbancefor each antibody is shown as a relative value to the absorbance of acontrol in the absence of antibody which is taken as 100.

FIG. 6 presents graphs showing a result of TLR4/MD-2 ELISA foranti-HMGB1 antibodies (HMG233, HMG236, HMG481, HMG487, and HMG446). Theabsorbance for each antibody is shown as a relative value to theabsorbance of a control in the absence of antibody which is taken as100.

FIG. 7 is a graph showing a time course of serum human HMGB1concentration in normal mice.

FIG. 8 is a graph showing a time course of serum anti-human HMGB1antibody concentration in normal mice.

FIG. 9 is a diagram showing that antigens can be eliminated from plasmaby using antibodies that bind to target antigens in a pH-dependentmanner and have FcRn-binding activity in a neutral pH range.

FIG. 10 is a diagram showing sensorgrams for 6RKE02-IgG1 to hsIL-6R atpH 7.4 and pH 6.0.

FIG. 11 is a graph showing a result of biological activity evaluationusing BaF3 cells expressing human gp130 (BaF/gp130).

FIG. 12 shows a time course of plasma hsIL-6R concentration afterantibody administration in an infusion test using normal mice.

FIG. 13 shows a protocol of in vivo drug efficacy test (Test 1) usingnormal mice and the SAA inhibitory effect (mean±SE) of antibodyadministration.

FIG. 14 is a diagram showing a protocol of in vivo drug efficacy test(Test 2) using normal mice and the SAA inhibitory effect (mean±SE) ofantibody administration.

FIG. 15 shows a non-limiting action mechanism for the elimination ofsoluble antigen from plasma by administering an antibody that binds toan antigen in an ion concentration-dependent manner and whose Fcγreceptor binding is enhanced at a neutral pH as compared to existingneutralizing antibodies.

FIG. 16 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1 whichbinds to human IL-6 receptor in a pH-dependent manner or H54/L28-IgG1.

FIG. 17 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1 whichbinds to human IL-6 receptor in a pH-dependent manner, Fv4-IgG1-F760which is an Fv4-IgG1 variant that lacks mouse FcγR binding,Fv4-IgG1-F1022 which is an Fv4-IgG1 variant with enhanced mouse FcγRbinding, or Fv4-IgG1-Fuc which is an Fv4-IgG1 antibody with low fucosecontent.

FIG. 18 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1 orantigen-binding molecules comprising as the heavy chain, VH3-IgG1-F1022or VH3-IgG1-F1093 which is a VH3-IgG1-F1022 variant with improved FcRnbinding in an acidic pH range.

FIG. 19 shows a concentration time course of the administeredantigen-binding molecules in the plasma of human FcRn transgenic miceadministered with Fv4-IgG1 or antigen-binding molecules comprising asthe heavy chain, VH3-IgG1-F1022 or VH3-IgG1-F1093 which is aVH3-IgG1-F1022 variant with improved FcRn binding in an acidic pH range.

FIG. 20 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1,Fv4-IgG1-F1087 which is an Fv4-IgG1 variant with enhanced mouse FcγRbinding (in particular, enhanced mouse FcγRIIb binding and mouse FcγRIIIbinding), and Fv4-IgG1-F1182 which is an Fv4-IgG1 variant with enhancedmouse FcγR binding (in particular, enhanced mouse FcγRI binding andmouse FcγRIV binding).

FIG. 21 shows a concentration time course of the administeredantigen-binding molecules in the plasma of human FcRn transgenic miceadministered with Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 andFv4-IgG1-F1412 which are Fv4-IgG1-F1087 variants with improved FcRnbinding in an acidic pH range.

FIG. 22 shows a concentration time course of the administeredantigen-binding molecules in the plasma of human FcRn transgenic miceadministered with Fv4-IgG1, Fv4-IgG1-F1182, and Fv4-IgG1-F1181 which isan Fv4-IgG1-F1182 variant with improved FcRn binding in an acidic pHrange.

FIG. 23 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1,Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and Fv4-IgG1-F1412 which areFv4-IgG1-F1087 variants with improved FcRn binding in an acidic pHrange.

FIG. 24 shows a time course of human IL-6 receptor concentration in theplasma of human FcRn transgenic mice administered with Fv4-IgG1,Fv4-IgG1-F1182, and Fv4-IgG1-F1181 which is an Fv4-IgG1-F1182 variantwith improved FcRn binding in an acidic pH range.

FIG. 25 shows a time course of human IL-6 receptor concentration in theplasma of normal mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44 whichis an Fv4-mIgG1 variant with enhanced mouse FcγRIIb binding and mouseFcγRIII binding, and Fv4-mIgG1-mF46 which is an Fv4-mIgG1 variant withfurther enhanced mouse FcγRIIb binding and mouse FcγRIII binding.

FIG. 26 shows a time course of human IL-6 receptor concentration in theplasma of FcγRIII-deficient mice administered with Fv4-mIgG1,Fv4-mIgG1-mF44 which is an Fv4-mIgG1 variant with enhanced mouse FcγRIIbbinding and mouse FcγRIII binding, and Fv4-mIgG1-mF46 which is anFv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouseFcγRIII binding.

FIG. 27 shows a time course of human IL-6 receptor concentration in theplasma of Fc receptor γ chain-deficient mice administered withFv4-mIgG1, Fv4-mIgG1-mF44 which is an Fv4-mIgG1 variant with enhancedmouse FcγRIIb binding and mouse FcγRIII binding, and Fv4-mIgG1-mF46which is an Fv4-mIgG1 variant with further enhanced mouse FcγRIIbbinding and mouse FcγRIII binding.

FIG. 28 shows a time course of human IL-6 receptor concentration in theplasma of FcγRIIb-deficient mice administered with Fv4-mIgG1,Fv4-mIgG1-mF44 which is an Fv4-mIgG1 variant with enhanced mouse FcγRIIbbinding and mouse FcγRIII binding, and Fv4-mIgG1-mF46 which is anFv4-mIgG1 variant with further enhanced mouse FcγRIIb binding and mouseFcγRIII binding.

FIG. 29 shows a result of evaluating the platelet aggregation ability ofthe omalizumab-G1d-v3/IgE immunocomplex by platelet aggregation assayusing platelets derived from donors with FcγRIIa allotype (R/H).

FIG. 30 shows a result of evaluating the platelet aggregation ability ofthe omalizumab-G1d-v3/IgE immunocomplex by platelet aggregation assayusing platelets derived from donors with FcγRIIa allotype (H/H).

FIG. 31 shows a result of assessing CD62p expression on the membranesurface of washed platelets. The black-filled area in the graphindicates a result of ADP stimulation after reaction with PBS. The areathat is not filled in the graph indicates a result of ADP stimulationafter reaction with the immunocomplex.

FIG. 32 shows a result of assessing the expression of active integrin onthe membrane surface of washed platelets. The black-filled area in thegraph indicates a result of ADP stimulation after reaction with PBS. Thearea that is not filled in the graph indicates a result of ADPstimulation after reaction with the immunocomplex.

FIG. 33 shows a graph in which the horizontal axis shows the relativevalue of FcγRIIb-binding activity of each PD variant, and the verticalaxis shows the relative value of FcγRIIa type R-binding activity of eachPD variant. The value for the amount of binding of each PD variant toeach FcγR was divided by the value for the amount of binding ofIL6R-F652 (SEQ ID NO: 162)/IL6R-L, which is a control antibody prior tointroduction of the alteration (IL6R-F652 is an antibody heavy chaincomprising an altered Fc with substitution of Pro at position 238 (EUnumbering) with Asp), to each FcγR; and then the obtained value wasmultiplied by 100, and used as the relative binding activity value foreach PD variant to each FcγR. The F652 plot in the figure shows thevalue for IL6R-F652/IL6R-L.

FIG. 34 shows a graph in which the vertical axis shows the relativevalue of FcγRIIb-binding activity of variants produced by introducingeach alteration into GpH7-B3 (SEQ ID NO: 168)/GpL16-k0 (SEQ ID NO: 169)which does not have the P238D alteration, and the horizontal axis showsthe relative value of FcγRIIb-binding activity of variants produced byintroducing each alteration into IL6R-F652 (SEQ ID NO: 162)/IL6R-L whichhas the P238D alteration. The value for the amount of FcγRIIb binding ofeach variant was divided by the value for the amount of FcγRIIb bindingof the pre-altered antibody; and then the obtained value was multipliedby 100, and used as the value of relative binding activity. Here, regionA contains alterations that exhibit the effect of enhancing FcγRIIbbinding in both cases where an alteration is introduced intoGpH7-B3/GpL16-k0 which does not have P238D and where an alteration isintroduced into IL6R-F652/IL6R-L which has P238D. Region B containsalterations that exhibit the effect of enhancing FcγRIIb binding whenintroduced into GpH7-B3/GpL16-k0 which does not have P238D, but do notexhibit the effect of enhancing FcγRIIb binding when introduced intoIL6R-F652/IL6R-L which has P238D.

FIG. 35 shows a crystal structure of the Fc (P238D)/FcγRIIbextracellular region complex.

FIG. 36 shows an image of superimposing the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex and the model structure ofthe Fc (WT)/FcγRIIb extracellular region complex, with respect to theFcγRIIb extracellular region and the Fc CH2 domain A by the leastsquares fitting based on the Cα atom pair distances.

FIG. 37 shows comparison of the detailed structure around P238D aftersuperimposing the crystal structure of the Fc (P238D)/FcγRIIbextracellular region complex and the model structure of the Fc(WT)/FcγRIIb extracellular region complex with respect to the only FcCH2 domain A or the only Fc CH2 domain B by the least squares fittingbased on the Cα atom pair distances.

FIG. 38 shows that a hydrogen bond can be found between the main chainof Gly at position 237 (EU numbering) in Fc CH2 domain A, and Tyr atposition 160 in FcγRIIb in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

FIG. 39 shows that an electrostatic interaction can be found between Aspat position 270 (EU numbering) in Fc CH2 domain B, and Arg at position131 in FcγRIIb in the crystal structure of the Fc (P238D)/FcγRIIbextracellular region complex.

FIG. 40 shows a graph in which the horizontal axis shows the relativevalue of FcγRIIb-binding activity of each 2B variant, and the verticalaxis shows the relative value of FcγRIIa type R-binding activity of each2B variant. The value for the amount of binding of each 2B variant toeach FcγR was divided by the value for the amount of binding of acontrol antibody prior to alteration (altered Fc with substitution ofPro at position 238 (EU numbering) with Asp) to each FcγR; and then theobtained value was multiplied by 100, and used as the value of relativebinding activity of each 2B variant towards each FcγR.

FIG. 41 shows Glu at position 233 (EU numbering) in Fc Chain A and thesurrounding residues in the extracellular region of FcγRIIb in thecrystal structure of the Fc (P238D)/FcγRIIb extracellular regioncomplex.

FIG. 42 shows Ala at position 330 (EU numbering) in Fc Chain A and thesurrounding residues in the extracellular region of FcγRIIb in thecrystal structure of the Fc (P238D)/FcγRIIb extracellular regioncomplex.

FIG. 43 shows the structures of Pro at position 271 (EU numbering) of FcChain B after superimposing the crystal structures of the Fc(P238D)/FcγRIIb extracellular region complex and the Fc (WT)/FcγRIIIaextracellular region complex by the least squares fitting based on theCα atom pair distances with respect to Fc Chain B.

FIG. 44 shows an image of the Fc (P208)/FcγRIIb extracellular regioncomplex determined by X-ray crystal structure analysis. For each of theCH2 and CH3 domains in the Fc portion, those on the left side arereferred to as domain A and those on the right side are referred to asdomain B.

FIG. 45 shows comparison after superimposing the structures of Fc(P208)/FcγRIIb extracellular region complex and Fc (WT)/FcγRIIaextracellular region complex (PDB code: 3RY6) determined by X-raycrystal structure analysis with respect to the CH2 domain A of the Fcportion by the least squares fitting based on the Cα atom pairdistances. In the diagram, the structure drawn with heavy line shows theFc (P208)/FcγRIIb extracellular region complex, while the structuredrawn with thin line indicates the structure of Fc (WT)/FcγRIIaextracellular region complex. Only the CH2 domain A of the Fc portion isdrawn for the Fc (WT)/FcγRIIa extracellular region complex.

FIG. 46 shows in the X-ray crystal structure of the Fc (P208)/FcγRIIbextracellular region complex, a detailed structure around Asp atposition 237 (EU numbering) in the CH2 domain A of the Fc portion, whichforms a hydrogen bond with Tyr at position 160 in FcγRIIb at the mainchain moiety.

FIG. 47 shows in the X-ray crystal structure of the Fc (P208)/FcγRIIbextracellular region complex, the structure of amino acid residuesaround Asp at position 237 (EU numbering) in the CH2 domain A of the Fcportion, which forms a hydrogen bond with Tyr at position 160 in FcγRIIbat the main chain moiety.

FIG. 48 shows comparison around the loop at positions 266 to 271 (EUnumbering) after superimposing the X-ray crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex shown in Reference Example15 and the Fc (P208)/FcγRIIb extracellular region complex with respectto the CH2 domain B of the Fc portion by the least squares fitting basedon the Cα atom pair distances. When compared to Fc (P238D), Fc (P208)has the H268D alteration at position 268 and the P271G alteration atposition 271 (EU numbering) in the loop.

FIG. 49 is a diagram showing the structure around Ser239 in the CH2domain B of the Fc portion in the X-ray crystal structure of the Fc(P208)/FcγRIIb extracellular region complex, along with the electrondensity determined by X-ray crystal structure analysis with 2Fo-Fccoefficient.

FIG. 50 shows comparison after superimposing the three-dimensionalstructures of the Fc (P208)/FcγRIIaR extracellular region complex and Fc(P208)/FcγRIIb extracellular region complex determined by X-ray crystalstructure analysis by the least squares fitting based on the Cα atompair distances.

FIG. 51 shows comparison around Asp at position 237 (EU numbering) inthe CH2 domain A of the Fc portion between the X-ray crystal structuresof the Fc (P208)/FcγRIIaR extracellular region complex and the Fc(P208)/FcγRIIb extracellular region complex, along with the electrondensity determined by X-ray crystal structure analysis with 2Fo-Fccoefficient.

FIG. 52 shows comparison around Asp at position 237 (EU numbering) inthe CH2 domain B of the Fc portion between the X-ray crystal structuresof the Fc (P208)/FcγRIIaR extracellular region complex and the Fc(P208)/FcγRIIb extracellular region complex, along with the electrondensity determined by X-ray crystal structure analysis with 2Fo-Fccoefficient.

FIG. 53 shows comparison between the constant-region sequences of G1dand G4d. In the diagram, the amino acids boxed with thick-frame indicatepositions with different amino acid residues between G1d and G4d.

FIG. 54 shows the structure of the heavy chain CDR3 of the 6RL#9antibody Fab fragment determined by X-ray crystal structure analysis.(i) shows the crystal structure of the heavy chain CDR3 obtained under acrystallization condition in the presence of calcium ion. (ii) shows thecrystal structure of the heavy chain CDR3 obtained under acrystallization condition in the absence of calcium ion.

FIG. 55 shows a time course of the plasma concentration of each antibodyin normal mice administered with antibody H54/L28-IgG1, FH4-IgG1, or6RL#9-IgG1.

FIG. 56 shows a time course of the plasma concentration of soluble humanIL-6 receptor (hsIL-6R) in normal mice administered with antibodyH54/L28-IgG1, FH4-IgG1, or 6RL#9-IgG1.

FIG. 57 shows ion-exchange chromatograms for an antibody having humanVk5-2 sequence and an antibody having h Vk5-2_L65 sequence which has analtered glycosylation sequence in the human Vk5-2 sequence. Solid lineindicates a chromatogram for an antibody having human Vk5-2 sequence(heavy chain: CIM_H (SEQ ID NO: 67); light chain: hVk5-2 (SEQ ID NO:57)); broken line indicates a chromatogram for an antibody havinghVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 67); light chain:hVk5-2_L65 (SEQ ID NO: 70)).

FIG. 58A shows ion-exchange chromatograms for an antibody havingLfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55); light chain:LfVk1_Ca (SEQ ID NO: 83)) and an antibody having a sequence in which Asp(D) in the LfVk1_Ca sequence is substituted with Ala (A) after storageat 5° C. (solid line) or 50° C. (dotted line). After storage at 5° C.,the highest peak in the chromatogram for each antibody is defined as amain peak, and the y axis of each ion-exchange chromatogram wasnormalized to the main peak. The graph shows a chromatogram for anantibody having LfVk1_Ca (SEQ ID NO: 83) as the light chain.

FIG. 58B shows a chromatogram for an antibody having LfVk1_Ca1 (SEQ IDNO: 85) as the light chain.

FIG. 58C shows a chromatogram for an antibody having LfVk1_Ca2 (SEQ IDNO: 86) as the light chain.

FIG. 58D shows a chromatogram for an antibody having LfVk1_Ca3 (SEQ IDNO: 87) as the light chain.

FIG. 59A shows ion-exchange chromatograms for an antibody havingLfVk1_Ca sequence (heavy chain: GC_H (SEQ ID NO: 55); light chain:LfVk1_Ca (SEQ ID NO: 83)) and an antibody having LfVk1_Ca6 sequence(heavy chain: GC_H (SEQ ID NO: 55); light chain: LfVk1_Ca6 (SEQ ID NO:88)) in which Asp (D) at position 30 (Kabat numbering) in the LfVk1_Casequence is substituted with Ser (S) after storage at 5° C. (solid line)or 50° C. (dotted line). After storage at 5° C., the highest peak in thechromatogram for each antibody is defined as a main peak, and the y axisof each ion-exchange chromatogram was normalized to the main peak. Thegraph shows a chromatogram for an antibody having LfVk1_Ca (SEQ ID NO:83) as the light chain.

FIG. 59B shows a chromatogram for an antibody having LfVk1_Ca6 (SEQ IDNO: 88) as the light chain.

FIG. 60 shows the relationship between designed amino acid distribution(indicated with “Design”) and amino acid distribution for sequenceinformation on 290 clones isolated from E. coli introduced with a genelibrary of antibodies that bind to antigens in a Ca-dependent manner(indicated with “Library”). The horizontal axis indicates amino acidposition (Kabat numbering). The vertical axis indicates percentage inamino acid distribution.

FIG. 61 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RC1IgG_(—)010, antibody 6RC1IgG_(—)012, and antibody6RC1IgG_(—)019 under a high calcium ion concentration (1.2 mM)condition. The horizontal axis shows time, and the vertical axis showsRU value.

FIG. 62 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RC1IgG_(—)010, antibody 6RC1IgG_(—)012, and antibody6RC1IgG_(—)019 under a low calcium ion concentration (3 μM) condition.The horizontal axis shows time, and the vertical axis shows RU value.

FIG. 63 shows the relationship between designed amino acid distribution(indicated with “Design”) and amino acid distribution for sequenceinformation on 132 clones isolated from E. coli introduced with a genelibrary of antibodies that bind to antigens in a pH-dependent manner(indicated with “Library”). The horizontal axis shows amino acidposition (Kabat numbering). The vertical axis indicates percentage inamino acid distribution.

FIG. 64 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RpH#01, antibody 6RpH#02, and antibody 6RpH#03 at pH 7.4. Thehorizontal axis shows time, and the vertical axis shows RU value.

FIG. 65 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RpH#01, antibody 6RpH#02, and antibody 6RpH#03 at pH 6.0. Thehorizontal axis shows time, and the vertical axis shows RU value.

MODE FOR CARRYING OUT THE INVENTION

The present invention provides antigen-binding molecules that reduce aplasma antigen concentration, which have the properties of (1) to (6)below:

-   -   (1) the antigen-binding molecules have an antigen-binding        domain(s) and a receptor-binding domain(s);    -   (2) the receptor-binding domain(s) has human neonatal Fc        receptor (FcRn)-binding activity under an acidic pH range        condition;    -   (3) the human Fc receptor-binding activity of the        receptor-binding domain(s) is stronger than that of native human        IgG under a neutral pH range condition;    -   (4) the antigen-binding activity of the antigen-binding        domain(s) changes depending on the ion concentration condition;    -   (5) the antigen (as a binding target) has two or more        physiological activities; and    -   (6) binding of the antigen-binding molecule inhibits one or more        of the physiological activities of the antigen (as a binding        target), while at least one physiological activity is        maintained.

In the present invention, the physiological activity is a general termfor activities that cause quantitative and/or qualitativechanges/influence in the living organism, tissue, cell, protein, DNA,RNA, or such; and for example, the activity of regulating a biologicalfunction such as metabolism, growth, reproduction, maintenance ofhomeostasis, mental activity, and biological defense. More specifically,it includes activities of regulating cell proliferation and maturation,metabolism mediated by the endocrine system, signal transduction in thenervous system, blood circulation, wound healing, immune response, andcell migration. Physiological activity can be reworded as biologicalactivity. A physiologically active substance refers to a substance withsuch physiological activities. Physiologically active substances exerttheir physiological activities by acting on specific molecules (targetmolecules) that constitute the living organism, and conferring certainchange/influence. In the present invention, physiologically activesubstances are also referred to as antigens that have physiologicalactivities. Physiologically active substances of the present inventionmay be any substances as long as they have physiological activities; butpreferably, they are polypeptides and modified products thereof thathave physiological activities (physiologically active peptides).Preferred target molecules for physiologically active substances toexert physiological activities are cell surface receptors andintracellular receptors. Physiologically active substances exert theirphysiological activities by binding to a specific receptor andtransducing signals into cells. When physiologically active substancesresult from conversion of precursors without physiological activitiesinto mature types that have physiological activities, enzymesresponsible for such conversion are also included in the physiologicallyactive substances of the present invention. In this case, targetmolecules are substrates (precursors) for the enzymes. Physiologicallyactive substances may be substances produced by organisms (humans ornonhuman organisms) or artificially synthesized substances.Physiologically active substances that might be a cause of disease inorganisms (preferably humans) are preferred in the present invention.

Examples of physiologically active peptides include cell growth factorsincluding fibroblast growth factors (FGFs), transforming growth factors(TGFs), bone morphogenetic factors (BMPs), epidermal growth factors(EGFs), platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF), nerve growth factor (NGF), vascular endothelial growthfactor (VEGF), hepatocyte growth factor (HGF), and bone morphogeneticfactor (BMP); cytokines including interferons (IFNs), interleukins(ILs), colony stimulation factors (CSFs), erythropoietin, and tumornecrosis factor (TNF); various hormones such as insulin and parathyroidhormone (PTH); enzymes including proteases such as matrixmetalloproteinases (MMPs); and enzyme inhibitory factors such as TIMPs(tissue inhibitor of metalloproteases).

Physiologically active substances of the present invention arepreferably physiologically active substances derived from mammals,especially preferably physiologically active substances derived fromhumans.

Physiologically active substances can be obtained, for example, bypurifying from a living organism. Alternatively, physiologically activesubstances can be prepared by chemical synthesis. When physiologicallyactive substances are physiologically active peptides, they can also beprepared as recombinant peptides by using genetic engineeringtechniques. Specifically, nucleic acids encoding physiologically activepeptides can be synthesized based on amino acid sequences of thephysiologically active peptides or nucleotide sequences encoding them,by gene cloning methods or nucleotide synthesis methods known to thoseskilled in the art. After the nucleic acids are inserted into knownexpression vectors to transform appropriate host cells, physiologicallyactive peptides of interest can be purified from the host cells orculture supernatants by known methods. Such purification can be achievedby using multiple chromatographies such as typical ion chromatographiesand affinity chromatographies once or several times in combination oralone. Furthermore, the physiologically active peptides may be preparedas partial peptides, which comprise a portion of a physiologicallyactive peptide, or may be prepared as fusion peptides by fusing withdifferent polypeptides such as peptide tags and Fc fragments, as long asthey retain their original physiological activities. Fusion peptides canbe prepared by fusing in frame genes encoding two or more desiredpolypeptide fragments and inserting the resulting fusion genes intoexpression vectors as described above (Sambrook J et al., MolecularCloning 2^(nd) ed. (1989) 9.47-9.58, Cold Spring Harbor Lab. Press).Target molecules that bind to physiologically active substances can alsobe obtained by similar methods.

Regarding methods for assaying in vitro physiological activity,physiologically active substances and their target molecules areprepared, and then their in vitro physiological activity can be assayedusing methods capable of detecting their binding, for example, ELISA,FACS, and Biacore. Alternatively, the physiological activities can bemeasured by detecting cellular changes (for example, changes in cellproliferation or morphology, and changes in gene or protein expression)after reacting physiologically active substances withreceptor-expressing cells. Meanwhile, in vivo physiological activitiescan be measured by observing changes in animals (for example, changesassociated with biological functions such as metabolism, growth,maintenance of homeostasis) after administering physiologically activesubstances to the animals.

In the present invention, “having two or more physiological activities”means that a physiologically active substance has the property ofbinding to two or more different target molecules. A physiologicallyactive substance may bind directly to target molecules, or may bindindirectly to target molecules after binding to a substance other thanthe target molecules that promotes binding to the target molecules. Adomain(s) responsible for target molecule binding exists in aphysiologically active substance. It is preferable that there aremultiple binding domains in a physiologically active substancecorresponding to two or more different target molecules. In particular,it is preferable that the binding domains are located in thethree-dimensional structure, at positions distant enough not to inhibitall the binding of the physiologically active substance to the targetmolecules at the same time when an antigen-binding molecule of thepresent invention binds to the physiologically active substance.

In the present invention, “having an activity” means that measuredvalues are greater than the background value (or a measured value for anegative control) in a system capable of measuring the activity. Forexample, having a binding activity means that measured values aregreater than the background value in a system capable of measuringbinding activity such as ELISA, FACS, and Biacore. In the presentinvention, the ratio of a measured value to the background value ispreferably twice or more, more preferably three times or more, stillmore preferably five times or more, and particularly preferably 10 timesor more.

In the present invention, “inhibiting activity” means that valuesmeasured in a system capable of measuring the activity after adding asubstance are lower than values measured before adding the substance (orthe measured value when a negative control is added). For example,inhibiting binding activity means that values measured after adding asubstance are lower than values measured before adding the substance ina system capable of measuring the binding activity such as ELISA, FACS,and Biacore. In the present invention, the ratio of a measured valueafter adding a substance to a measured value before adding the substance(or a measured value when adding a negative control) is preferably 80%or less, more preferably 50% or less, still more preferably 30% or less,and particularly preferably 10% or less.

In the present invention, “maintaining an activity” means that ameasured value after adding a substance is 80% or more of a measuredvalue before adding the substance (or a measured value when a negativecontrol is added) in a system capable of measuring the activity. Forexample, maintaining a binding activity means that a measured valueafter adding a substance is 80% or more of a measured value beforeadding the substance in a system capable of measuring the bindingactivity such as ELISA, FACS, and Biacore. In the present invention, theratio of a measured value after adding a substance to a measured valuebefore adding the substance (or a measured value when a negative controlis added) is preferably 85% or more, more preferably 90% or more, andstill more preferably 95% or more.

In the present invention, “reducing plasma antigen concentration” meansthat plasma antigen concentration when an antigen-binding molecule ofthe present invention is administered to a subject is lower compared towhen a negative control is administered to a subject. The percentage ofreduction is not particularly limited; however, the percentage ispreferably 80% or less, more preferably 50% or less, still morepreferably 30% or less, and most preferably 10% or less. Furthermore, inthe present invention, “reducing antigen concentration in plasma” isreworded as “promoting antigen elimination from plasma (clearance)”,“shortening antigen retention time in plasma”, and “shortening antigenhalf-life in plasma”. Meanwhile, “in plasma” may also be “in serum”.Antigen-binding molecules provided by the present invention can beadministered to subjects (a living organism) by, for example,intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal,parenteral, or intramuscular injection. Subjects to whichantigen-binding molecules of the present invention are administered arepreferably animals, more preferably mammals, and still more preferablyhumans.

The reduction of antigen concentration may be achieved by promotingantigen uptake into cells. Meanwhile, the reduction of antigenconcentration preferably results in a decrease of the physiologicalactivity of the antigen in vivo, and particularly preferably results ina decrease of all the physiological activities of the antigen.

Antigen concentration can be measured by appropriately using methodsknown to those skilled in the art. When an antigen is a physiologicallyactive peptide, the concentration of the physiologically active peptidein a sample of unknown concentration can be determined by preparingstandard samples of known concentration and a system that canquantitatively determine their concentration (for example, ELISA orBiacore), and creating a standard curve that shows the relationshipbetween measured values and respective concentrations. Samples includeplasma from the living organism, cell culture media, and cell extracts.

In the present invention, antigen uptake into cells means that theantigen is incorporated into cells by endocytosis. Whether antigenuptake into cells is promoted can be assessed by, for example, testingwhether the antigen concentration in the culture medium is decreased ascompared to a control or whether the antigen concentration in cells isincreased as compared to a control after the antigen is added to thecell culture medium. Promotion of antigen uptake into cells meanspromotion of antigen elimination from plasma in the living organism.Thus, whether antigen uptake into cells is promoted can be assessed by,for example, testing whether the antigen concentration in plasma isdecreased as compared to a control after the antigen is administered toa living organism.

Antigen-binding molecules provided by the present invention are notparticularly limited as long as they have the properties described in(1) to (6) above. However, they are preferably polypeptides having theproperty of binding specifically to an antigen, human FcRn, and human Fcreceptor; and they are more preferably antibodies, and particularlypreferably IgGs. Antibodies may be chimeric antibodies, humanizedantibodies, human antibodies, and such. Antibodies may also bebispecific antibodies, antibody modification products to which varioustypes of molecules are linked, polypeptides comprising antibodyfragments, and such. The antigen-binding molecules provided by thepresent invention comprise an antigen-binding domain(s) and areceptor-binding domain(s). The above-described domain refers toconstitutional units that can be divided in parts and isolated. The sizeof the domain is not particularly limited. Each domain comprises apolypeptide. The antigen-binding domain of the present invention is notparticularly limited as long as it has the property of bindingspecifically to an antigen; however, preferred examples includeantibodies and fragments thereof (variable region, Fab, F(ab′)2, Fv,CDR, etc.), antibody-like molecules referred to as scaffold (DARPins(WO2002/020565), Affibody (WO1995/001937), Avimer (WO2004/044011;WO2005/040229), Adnectin (WO2002/032925), etc.), and target molecules(receptors) and fragments thereof (soluble receptors), which bind tophysiologically active substances. Particularly preferred examplesinclude antibody variable regions. Meanwhile, receptor-binding domainsof the present invention are not particularly limited as long as theyhave the property of binding specifically to human FcRn and/or human Fcreceptor; however, preferred examples include antibodies (IgGs) andfragments thereof (the constant region, Fc, etc.), albumin and fragmentsthereof (domain 3), anti-FcRn antibody and fragments thereof (variableregion, Fab, F(ab′)2, Fv, CDR, etc.), anti-FcRn antibody-like molecules(DARPins (WO2002/020565), Affibody (WO 1995/001937), Avimer(WO2004/044011; WO2005/040229), Adnectin (WO2002/032925), etc.), andanti-FcRn peptides. More preferred examples include IgG Fc regions.Human Fc receptors of the present invention are not particularly limitedas long as human IgG, in particular human IgG Fc region, binds to them;however, preferably they are human FcRn and human Fcγ receptors. IgGsmay be derived from nonhuman animals or humans; however, IgGs arepreferably human IgGs, particularly preferably human IgG1. As describedbelow, it is preferable that the Fc region of the IgGs has amino acidalterations.

An antigen-binding molecule of the present invention may comprise atleast one receptor-binding domain. For example, a single antigen-bindingmolecule may comprise one antigen-binding domain and onereceptor-binding domain, or may comprise one antigen-binding domain andmultiple receptor-binding domains. When a single antigen-bindingmolecule comprises multiple receptor-binding domains, all of thereceptor-binding domains may bind to the same type of human Fcreceptors, or each of the domains may bind to different types of humanFc receptors. On the other hand, since one of the requirements forantigen-binding molecules of the present invention to fulfill is to havehuman FcRn-binding activity, it is preferable that at least onereceptor-binding domain comprised in an antigen-binding molecule of thepresent invention binds to human FcRn. Without particular limitation,embodiments where a single antigen-binding molecule comprises tworeceptor-binding domains include antigen-binding molecules of which tworeceptor-binding domains both bind to human FcRn, and antigen-bindingmolecules of which one of the receptor-binding domains binds to humanFcRn and the other binds to human Fcγ receptor. Alternatively, when asingle antigen-binding molecule comprises one receptor-binding domain,the receptor-binding domain may at least bind to human FcRn and may havethe property that the single domain simultaneously binds to other typesof human Fc receptors. Such receptor-binding domains include, forexample, IgG Fc regions. IgG Fc regions have the property of binding tohuman FcRn and human Fcγ receptor.

Herein, native human IgG refers to naturally-occurring human IgG. It isdesirable that a fucose-containing sugar chain is linked at position 297(EU numbering) in the Fc region. As a native human IgG,naturally-occurring human IgG1, IgG2, IgG3, and IgG4 can be used.Preferred native human IgG is a naturally-occurring human IgG1. Whethera linked sugar chain is a fucose-containing sugar chain can be assessed,for example, by the method described below. Test human IgG is incubatedwith N-Glycosidase F (Roche diagnostics) to release its sugar chains(Weitzhandler et al., J. Pharma. Sciences (1994) 83(12): 1670-1675).Then, the reaction solution is deproteinated by reacting ethanol (Schenket al., J. Clin. Investigation (2001) 108 (11): 1687-1695) andconcentrated to dryness, followed by fluorescence labeling with2-aminopyridin (Bigge et al., Anal. Biochem. (1995) 230 (2): 229-238).Solid extraction is performed using cellulose cartridge to remove thereagents, and the resulting fluorescently 2-AB-labeled sugar chains areanalyzed by normal phase chromatography. Whether the sugar chain linkedto the Fc region of the test human IgG is a fucose-containing sugarchain can be assessed by observing peaks detected in the chromatogram.

“Chimeric antibodies” are antibodies prepared by combining sequencesderived from different animals. Specifically, the chimeric antibodyincludes, for example, antibodies having heavy and light chain variable(V) regions from a mouse antibody and heavy and light chain constant (C)regions from a human antibody.

“Humanized antibodies”, also referred to as reshaped human antibodies,are antibodies in which the complementarity determining regions (CDRs)of an antibody derived from a nonhuman mammal, for example, a mouse, aregrafted into the CDRs of a human antibody. Methods for identifying CDRsare known (Kabat et al., Sequence of Proteins of Immunological Interest(1987), National Institute of Health, Bethesda, Md.; Chothia et al.,Nature (1989) 342: 877). General genetic recombination technologiessuitable for grafting CDRs are also known (see European PatentApplication EP 125023; and WO96/02576).

“A bispecific antibody” refers to an antibody that has two variableregions in the same antibody molecule that recognize different epitopes.A bispecific antibody may be an antibody that recognizes two or moredifferent antigens, or an antibody that recognizes two or more differentepitopes on a same antigen.

Furthermore, polypeptides comprising antibody fragments include, forexample, Fab fragments, F(ab′)2 fragments, scFvs (Nat. Biotechnol. 2005September; 23(9): 1126-36), domain antibodies (dAbs) (WO2004/058821;WO2003/002609), scFv-Fc (WO2005/037989), dAb-Fc, and Fc fusion proteins.When a molecule contains an Fc region, the Fc region can be used as thereceptor-binding domain. The Fc region refers to a portion of the heavychain constant region, which starts from the N terminus of the hingeregion corresponding to the papain cleavage site in the antibodymolecule and contains the hinge, and the CH2 and CH3 domains. The IgG Fcregion refers to, for example, from cysteine at position 226 (EUnumbering) up to the C terminus, or from proline at position 230 (EUnumbering) up to the C terminus; but are not limited thereto. Withoutparticular limitation, examples of the IgG Fc region include the Fcregions of human IgG1 (SEQ ID NO: 49), IgG2 (SEQ ID NO: 50), IgG3 (SEQID NO: 51), and IgG4 (SEQ ID NO: 52). The IgG Fc region is preferablythe Fc region of human IgG1.

Antibody-like molecule (scaffolding molecule or scaffold molecule) is ageneral name for molecules that have a common backbone structure and theproperty of being able to specifically bind to an antigen (CurrentOpinion in Biotechnology 2006, 17: 653-658; Current Opinion inBiotechnology 2007, 18: 1-10; Current Opinion in Structural Biology1997, 7: 463-469; Protein Science 2006, 15: 14-27). Antibody-likemolecules include, for example, DARPins (WO2002/020565), affibody(WO1995/001937), avimer (WO2004/044011; WO2005/040229), and adnectin(WO2002/032925).

Antibodies may contain modified sugar chains. Antibodies with modifiedsugar chains include, for example, antibodies with modifiedglycosylation (WO99/54342), antibodies that are deficient in sugarchain-attached fucose (WO00/61739; WO02/31140; WO2006/067847;WO2006/067913), and antibodies having sugar chains with bisecting GlcNAc(WO02/79255).

The binding between an antigen and an antigen-binding molecule of thepresent invention, or between a target molecule and an antigen withphysiological activities can be measured by using methods known to thoseskilled in the art such as ELISA, FACS, and Biacore. By setting themeasurement condition to an extracellular condition or intracellularcondition, differences in the binding activity under such conditions canbe assessed. Furthermore, such methods can be combined with theabove-described methods for measuring the physiological activity ofphysiologically active substances to assess whether the binding of anantigen-binding molecule of the present invention to an antigen withphysiological activities inhibits the physiological activity/activitiesof the antigen or maintains the physiological activity/activities.

The present invention provides antigen-binding molecules characterizedin that they inhibit one or more binding activities of an antigen to itstarget molecules by binding to the antigen while allowing the antigen tomaintain the binding activity to at least one type of its targetmolecules. Specifically, it is preferable that one or more physiologicalactivities of an antigen having two or more physiological activities areinhibited by binding of an antigen-binding molecule of the presentinvention to the antigen Inhibiting one or more physiological activities(or, are inhibited) means inhibiting the activity of an antigen, whichhas binding activity to multiple target molecules, to bind to one ormore types of its target molecules (or, are inhibited). Furthermore,when an antigen-binding molecule of the present invention binds to anantigen having two or more types of physiological activities, it ispreferable that the antigen maintains at least one type of physiologicalactivity among its physiological activities (or, at least one type ofphysiological activity is maintained). Maintaining at least one type ofphysiological activity (or is maintained) means that an antigenmaintains its binding-activity to at least one type of the targetmolecules (or is maintained) among the binding activities of the antigento multiple target molecules.

When an antigen with physiological activity that is present in excess ina living organism causes a disease, molecules, for example, neutralizingantibodies which inhibit the physiological activity by binding to theantigen, are expected to be useful in treating the disease. However,when the antigen has two or more types of physiological activities,neutralizing antibodies can inhibit only a single type of physiologicalactivity. On the other hand, antigen-binding molecules of the presentinvention can ultimately reduce the in vivo physiological activities bypromoting antigen elimination from blood (serum or plasma) even when theantigen maintains at least one type of physiological activity. Thus, theantigen-binding molecules of the present invention are very useful ascompared to common neutralizing antibodies.

In the present invention, when a human Fc receptor is human FcRn, thereceptor-binding domain of antigen-binding molecules is preferably anIgG Fc region, more preferably an Fc region variant in which at leastone amino acid in the IgG Fc region is altered. The IgG may be derivedfrom nonhuman animals or humans; however, the IgG is preferably humanIgG (IgG1, IgG2, IgG3, or IgG4), and particularly preferably human IgG1.Examples of amino acid alterations include amino acid substitution,insertion, and deletion; however, amino acid substitution is preferred.The number of amino acids to be altered is not particularly limited, andamino acids may be altered at only one site or at two or more sites. Insuch amino acid alteration, amino acids at any positions may be alteredto any amino acids as long as the Fc region variant after alteration hashuman FcRn-binding activity under acidic and neutral pH rangeconditions, and the human FcRn-binding activity under a neutral pH rangecondition is greater than that of human IgG. It is known that in aliving organism, generally the extracellular pH (for example, in plasma)is neutral and the intracellular pH (for example, in the endosome) isacidic. It is also known that the binding between IgG and FcRn isdetected only under an acidic (intracellular) pH condition and is almostundetectable under a neutral (extracellular) pH condition. For theantigen-binding molecules of the present invention, acidic pH ispreferably endosomal pH and neutral pH is preferably plasma pH.

If the receptor-binding domains of antigen-binding molecules provided bythe present invention can be conferred with the property of having humanFcRn-binding activity under intracellular and extracellular pHconditions and the property that the human FcRn-binding activity underan extracellular pH condition is stronger than that of human IgG,extracellularly antigen-bound antigen-binding molecules of the presentinvention bind to cell-surface FcRn and are internalized into cells,which results in promotion of antigen uptake into cells from the outsideof the cells. When administered to a living organism, suchantigen-binding molecules can reduce plasma antigen concentration anddecrease the physiological activities of antigens in vivo. Thus,antigen-binding molecules provided by the present invention are useful.

Human FcRn is structurally similar to major histocompatibility complex(MHC) class I polypeptides, and shares 22 to 29% sequence identity withclass I MHC molecules (Ghetie et al., Immunol. Today (1997) 18 (12),592-598). FcRn is expressed as a heterodimer consisting of β2microglobulin which is a soluble β chain (or light chain), andtransmembrane a chain (or heavy chain). FcRn α chain comprises threeextracellular domains (α1, α2, and α3). The α1 and α2 domains interactwith the FcRn-binding domain of antibody Fc region (Raghavan et al.,Immunity (1994) 1, 303-315).

The gene and amino acid sequences for human FcRn are registered underGenBank accession number NM_(—)001136019 (SEQ ID NO: 16) andNP_(—)001129491 (SEQ ID NO: 17), respectively. In addition to human, thegene and amino acid sequences for mouse FcRn are registered underGenBank accession number NM_(—)010189 (SEQ ID NO: 18) and NP_(—)034319(SEQ ID NO: 19), respectively; the gene and amino acid sequences for ratFcRn are registered under GenBank accession number NM_(—)033351 (SEQ IDNO: 20) and NP_(—)203502 (SEQ ID NO: 21), respectively.

Human FcRn (SEQ ID NO: 17) forms a complex with human β2 microglobulin(SEQ ID NO: 38) in vivo. The complex of soluble human FcRn and β2microglobulin can be produced using general recombination/expressionmethods. Such a soluble human FcRn/β2 microglobulin complex can be usedto assess the binding activity of receptor-binding domains of thepresent invention. In the present invention, unless otherwise specified,human FcRn refers to a form capable of binding to receptor-bindingdomains of the present invention, and includes, for example, a complexof human FcRn and human P2 microglobulin.

Receptor-binding domains that bind to FcRn under a neutral pH rangecondition more strongly than native human IgG can be produced byaltering the amino acids of human IgG Fc region. Examples of alterationinclude substitution, insertion, and deletion of one or more aminoacids. Alternatively, it is possible to use an antigen-binding domaincharacterized in binding to FcRn as the receptor-binding domain. Whetherthe FcRn-binding activity of a receptor-binding domain is higher thanthat of the native human IgG Fc region can be appropriately assessed bythe above-described methods.

In the present invention, the human FcRn-binding activity under anacidic pH range condition means the human FcRn-binding activity at pH4.0 to pH 6.5, preferably at pH 5.0 to pH 6.5, more preferably at any ofpH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, andparticularly preferably at pH 5.8 to pH 6.0 which is close to the pH ofearly endosome in vivo. Meanwhile, in the present invention, the humanFcRn-binding activity under a neutral pH range condition refers to humanFcRn-binding activity at pH 6.7 to pH 10.0, preferably at pH 7.0 to pH9.0, more preferably at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, and 8.0, and particularly preferably at pH 7.4 which isclose to the pH of plasma in vivo.

When the binding affinity between a receptor-binding domain and humanFcRn is very low at pH 7.4 and is difficult to determine accurately, thepH can be 7.0 instead of pH 7.4. Regarding the measurement temperature,the binding affinity between a receptor-binding domain and human FcRnmay be measured at any temperature between 10° C. and 50° C. The bindingaffinity between a receptor-binding domain and human FcRn is preferablydetermined at 15° C. to 40° C., more preferably at any temperature of20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35° C.Without particular limitation, the 25° C. temperature is a preferredembodiment.

As a receptor-binding domain that binds to human FcRn, an IgG Fc regionis altered preferably at sites including, for example, amino acids atpositions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 260, 262 to272, 274, 276, 278 to 289, 291 to 320, 324 to 341, 343, 345, 360 to 362,370, 375 to 378, 380, 382, 384 to 387, 389 to 391, 396, 413, 414, 416,422, 423, 424, 426 to 438, 440, and 442 (EU numbering). Morespecifically, such alteration includes, for example, alteration of aminoacids shown in Table 1. Such alteration can be used to enhance the humanFcRn binding of an IgG Fc region under a neutral pH range condition.

TABLE 1 POSITION AMINO ACID ALTERATION 256 P 280 K 339 T 385 H 428 L 434W, Y, F, A, H

Meanwhile, an example of alteration that can enhance the human FcRnbinding under an acidic pH range condition as compared to native humanIgG is shown in Table 2. From among such alterations, appropriatealterations that can also enhance the human FcRn binding under a neutralpH range condition can be selected and used in the present invention. Inalteration of IgG Fc region, particularly preferred sites for alterationinclude, for example, amino acid(s) at position(s) 234, 235, 236, 237,238, 239, 244, 245, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 260, 262, 265, 267, 270, 272, 274, 279, 280, 282, 283, 284, 285,286, 288, 289, 293, 295, 297, 298, 303, 305, 307, 308, 309, 311, 312,313, 314, 315, 316, 317, 318, 325, 326, 327, 328, 329, 330, 332, 334,338, 339, 340, 341, 343, 345, 360, 361, 362, 375, 376, 377, 378, 380,382, 384, 385, 386, 387, 389, 390, 391, 413, 422, 423, 424, 427, 428,430, 431, 433, 434, 435, 436, 437, 438, 440, and 442 (EU numbering).Furthermore, preferred site(s) besides those described above include,for example:

amino acid(s) at position(s) 252, 254, 256, 309, 311, 315, 433, and/or434 (EU numbering) and, in combination with those described above, aminoacid(s) at position(s) 253, 310, 435, and/or 426 (EU numbering), whichare described in WO1997/034631;amino acid(s) at position(s) 238, 252, 253, 254, 255, 256, 265, 272,286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376,378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439,and/or 447 (EU numbering), which are described in WO2000/042072;amino acid(s) at position(s) 251, 252, 254, 255, 256, 308, 309, 311,312, 385, 386, 387, 389, 428, 433, 434, and/or 436 (EU numbering), whichare described in WO2002/060919; amino acid(s) at position(s) 250, 314,and 428 (EU numbering), which are described in WO2004/092219;amino acid(s) at position(s) 238, 244, 245, 249, 252, 256, 257, 258,260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316,317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427,430, 431, 434, 436, 438, 440, and/or 442 (EU numbering), which aredescribed in WO2006/020114; andamino acid(s) at position(s) 251, 252, 307, 308, 378, 428, 430, 434,and/or 436 (EU numbering), which are described in WO2010/045193. Atleast one amino acid selected from the amino acids described above canbe altered to increase the human FcRn-binding activity under a neutralpH range condition. The number of amino acids to be altered is notparticularly limited, and amino acids may be altered at only one site ortwo or more sites.

TABLE 2 POSITION AMINO ACID ALTERATION 221 Y, K 222 Y 223 E, K 224 Y, E225 E, K, W 227 K, E, G 228 Y, K, G 230 E, G 232 K 233 R, S, M, T, W, Y,G 234 H, R, E, I, V, F, D, Y, G 235 Y, V, N, S, T, Q, D 236 I, V, K, P,E, Q, H, W, Y, D, T, M, A, F, S, N, R 237 I, W, S, T, E, R, N, Q, K, H,D, P, L, M 238 A, L, D, S, T, H, W, V, I, G, M, F, E, K 239 M, R, T, G,V, E, D, L, A 240 I, M, T 241 E, W, L 243 E, W 244 L 245 R 246 Y, H 247D 248 Y 249 P, Q, Y, H 250 I, E, Q 251 T, D 252 Y, W, Q 254 H 255 E, Y,H 256 A 257 A, I, M, N, S, V, T, L, Y, C 258 D, Y, H, A 259 I, F, N 260S, D, E, H, Y 262 L, E 263 I 264 F, A, I, T, N, S, D 265 R, P, G, A 266I 267 K, E, A 268 E, M 269 M, W, K, P, I, S, G, V, F, Y, A 270 K, S, I,A 271 A, V, S, Y, I, T 272 A, L, R, I, D, H, V, W, Y, P, T 274 M, F, G,E, I, T, N 276 D, F, H, R, L, V, W, A 278 R, S, V, M, N, I, L, D 279 A,D, G, H, M, N, Q, R, S, T, W, Y, C, I 281 D, Y 282 G, K, E, Y 283 A, D,F, G, H, I, K, L, N, P, Q, R, S, T, W, Y 284 T, L, Q, E 285 N, Y, W, Q,K, E, D, Y 286 F, L, Y, E, P, D, K, A 287 S, H 288 N, P, Y, H, D, I, V,C, E, G, L, Q, R 289 H 291 Q, H 292 Y, E, D 293 V 294 I, K, G 295 V, T296 E, I, L 298 F, E, T, H 299 W, F, H, Y 300 K, A, G, V, M, Q, N, E 301E 302 I 303 Y, E, A 304 N, T 305 A, H 306 Y 307 A, E, M, G, Q, H 308 A,R, F, C, Y, W, N, H 311 A, I, K, L, M, V, W, T, H 312 A, P, H 315 T, H316 K 317 A, P, H 318 N, T, R, L, Y 319 L, I, W, H, M, V, A 320 L, W, H,N 324 T, D 325 F, M, D 326 A 327 D, K, M, Y, H, L 328 G, A, W, R, F 329K, R, W 330 G, W, V, P, H, F 331 L, F, Y 332 F, H, K, L, M, R, S, W, T,Q, E, Y, D, N, V 333 L, F, M, A 334 A 335 H, F, N, V, M, W, I, S, P, L336 E, K 337 A 338 A 339 N, W 341 P 343 E, H, K, Q, R, T, Y 360 H, A 362A 375 R 376 A, G, I, M, P, T, V 377 K 378 Q, D, N, W 380 A, N, S, T, Q,R, H 382 A, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y 385 N, E 386 H 387H, Q 414 A 423 N 424 A 426 H, L, V, R 427 N 428 F 429 Q 430 A, F, G, H,I, K, L, M, N, Q, R, S, T, V, Y 431 H, K 432 H 433 P 434 G, T, M, S, 435K 436 I, L, T 437 H 438 K, L, T, W 440 K 442 K

Meanwhile, receptor-binding domains that originally have humanFcRn-binding activity under acidic pH range and neutral pH rangeconditions include, for example, an IgG Fc region, wherein amino acidsof the Fc region are selected from:

according to EU numbering,the amino acid at position 234 which is Arg;the amino acid at position 235 which is Gly, Lys, or Arg;the amino acid at position 236 which is Ala, Asp, Lys, or Arg;the amino acid at position 237 which is Lys, Met, or Arg;the amino acid at position 238 which is Ala, Asp, Lys, Leu, or Arg;the amino acid at position 239 which is Asp or Lys;the amino acid at position 244 which is Leu;the amino acid at position 245 which is Arg;the amino acid at position 248 which is Ile or Tyr;the amino acid at position 249 which is Pro;the amino acid at position 250 which is Ala, Glu, Phe, Ile, Met, Gln,Ser, Val, Trp, Gly, His, Leu, Asn, or Tyr;the amino acid at position 251 which is Arg, Asp, Glu, or Leu;the amino acid at position 252 which is Phe, Ser, Thr, Trp, or Tyr;the amino acid at position 253 which is Val;the amino acid at position 254 which is Ala, Gly, His, Ile, Gln, Ser,Val, or Thr;the amino acid at position 255 which is Ala, Asp, Phe, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Gly, Ser, Trp, Tyr, or Glu;the amino acid at position 256 which is Ala, Asp, Glu, Arg, Asn, Pro,Thr, Ser, or Gln;the amino acid at position 257 which is Ala, Gly, Ile, Leu, Met, Asn,Ser, Thr, or Val;the amino acid at position 258 which is Asp or His;the amino acid at position 260 which is Ser;the amino acid at position 262 which is Leu;the amino acid at position 265 which is Ala;the amino acid at position 267 which is Met or Leu;the amino acid at position 270 which is Lys or Phe;the amino acid at position 272 which is Ala, Leu, or Arg;the amino acid at position 274 which is Ala;the amino acid at position 279 which is Leu, Ala, Asp, Gly, His, Met,Asn, Gln, Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 280 which is Ala, Gly, His, Lys, Asn, Gln,Arg, Ser, Thr, or Glu;the amino acid at position 282 which is Ala or Asp;the amino acid at position 283 which is Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 284 which is Lys;the amino acid at position 285 which is Asn;the amino acid at position 286 which is Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or Glu;the amino acid at position 288 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Gln, Arg, Val, Trp, Tyr, or Ser;the amino acid at position 289 which is His;the amino acid at position 293 which is Val;the amino acid at position 295 which is Met;the amino acid at position 297 which is Ala;the amino acid at position 298 which is Gly;the amino acid at position 303 which is Ala;the amino acid at position 305 which is Ala or Thr;the amino acid at position 307 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;the amino acid at position 308 which is Ala, Phe, Ile, Leu, Met, Pro,Gln, or Thr;the amino acid at position 309 which is Ala, Asp, Glu, Pro, His, or Arg;the amino acid at position 311 which is Ala, His, Glu, Lys, Leu, Met,Ser, Val, Trp, or Ile;the amino acid at position 312 which is Ala, Asp, Pro, or His;the amino acid at position 313 which is Tyr or Phe;the amino acid at position 314 which is Ala, Leu, Lys, or Arg;the amino acid at position 315 which is Ala, Asp, Glu, Phe, Gly, Ile,Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or His;the amino acid at position 316 which is Ala, Glu, Phe, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Asp;the amino acid at position 317 which is Ala or Pro;the amino acid at position 318 which is Asn or Thr;the amino acid at position 325 which is Ala, Gly, Met, Leu, Ile, or Ser;the amino acid at position 326 which is Asp;the amino acid at position 327 which is Gly;the amino acid at position 328 which is Arg, Asp, Glu, or Tyr;the amino acid at position 329 which is Lys or Arg;the amino acid at position 330 which is Leu;the amino acid at position 332 which is Glu, Phe, His, Lys, Leu, Met,Arg, Ser, Trp, or Val;the amino acid at position 334 which is Leu;the amino acid at position 338 which is Ala;the amino acid at position 339 which is Asn, Thr, or Trp;the amino acid at position 340 which is Ala;the amino acid at position 341 which is Pro;the amino acid at position 343 which is Glu, His, Lys, Gln, Arg, Thr, orTyr;the amino acid at position 345 which is Ala;the amino acid at position 360 which is His;the amino acid at position 361 which is Ala;the amino acid at position 362 which is Ala;the amino acid at position 375 which is Ala or Arg;the amino acid at position 376 which is Ala, Gly, Ile, Met, Pro, Thr, orVal;the amino acid at position 377 which is Lys;the amino acid at position 378 which is Asp, Asn, or Val;the amino acid at position 380 which is Ala, Asn, Thr, or Ser;the amino acid at position 382 which is Ala, Phe, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Trp, Tyr, or Val;the amino acid at position 384 which is Ala;the amino acid at position 385 which is Ala, Gly, Lys, Ser, Thr, Asp,His, or Arg;the amino acid at position 386 which is Arg, Asp, Ile, Met, Ser, Thr,Lys, or Pro;the amino acid at position 387 which is Ala, Arg, His, Pro, Ser, Thr, orGlu;the amino acid at position 389 which is Ala, Asn, Pro, or Ser;the amino acid at position 390 which is Ala;the amino acid at position 391 which is Ala;the amino acid at position 413 which is Ala;the amino acid at position 423 which is Asn;the amino acid at position 424 which is Ala or Glu;the amino acid at position 427 which is Asn;the amino acid at position 428 which is Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 430 which is Ala, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr;the amino acid at position 431 which is His or Asn;the amino acid at position 433 which is Arg, Gln, His, Ile, Pro, Ser, orLys;the amino acid at position 434 which is Ala, Phe, Gly, Met, His, Ser,Trp, or Tyr;the amino acid at position 435 which is Lys, Arg, or Asn;the amino acid at position 436 which is Ala, His, Ile, Leu, Glu, Phe,Gly, Lys, Met, Asn, Arg, Ser, Thr, Trp, or Val;the amino acid at position 437 which is Arg;the amino acid at position 438 which is Lys, Leu, Thr, or Trp;the amino acid at position 440 which is Lys; andthe amino acid at position 442 which is Lys.

Amino acid positions to be selected may be only one, or two or morepositions. Combinations of amino acids of two or more positions include,for example, those described in Tables 3, 4-1 to 4-5, and 13-1 to 13-14.

TABLE 3 COMBINATION OF AMINO ACID ALTERATION M252Y/S254T/T256EM252Y/S254T/T256E/H433K/N434F/Y436H H433K/N434F/Y436H T307A/E380AT307A/E380A/N434H T307A/E380A/N434A N434H/N315H N434H/T289HN434H/T370A/E380A T250Q/M428L T250Q/N434A M252W/N434A M252Y/N434AT256A/N434A T256D/N434A T256E/N434A T256S/N434A P257I/Q311I T307A/N434AT307E/N434A T307Q/N434A V308P/N434A L309G/N434A Q311H/N434A Q311R/N434AN315D/N434A A378V/N434A E380S/N434A E382V/N434A S424E/N434A M428L/N434AN434A/Y436I T437Q/N434A T437R/N434A

TABLE 4-1 COMBINATION OF AMINO ACID ALTERATION L234I/L235DG236A/V308F/I332E G236R/L328R G236A/I332E/N434S S239E/V264I/A330Y/I332ES239E/V264I/I332E S239E/V264I/S298A/A330Y/I332E S239D/D265H/N297D/I332ES239D/E272Y/I332E S239D/E272S/I332E S239D/E272I/I332E S239D/N297D/I332ES239D/K326T/I332E S239Q/I332Q S239Q/I332N S239D/I332D S239D/I332ES239Q/I332E S239E/I332E F241W/F243W F241Y/F243Y/V262T/V264TF241W/F243W/V262A/V264A F241L/V262I F243L/V262I/V264WF243L/K288D/R292P/Y300L/V305I/P396L/H435K F243L/K288D/R292P/Y300L/H435KF243L/R292P/Y300L/V305I/P396L/H435K P245G/V308F T250I/V259I/V308FT250I/V308F T250I/V308F/N434S T250Q/V308F/M428L T250Q/M428L L251I/N434SL251N/N434S L251F/N434S L251V/N434S L251M/N434S T252L/T254S/T256FM252Y/S254T/T256E/N434M M252Y/S254T/T256E/M428L/N434S M252Y/S254T/T256EM252Y/S254T/T256E/V308F M252Y/S254T/T256E/N434S M252Y/S254T/T256E/N434AM252Y/S254T/T256E/M428L M252Y/S254T/T256E/T307Q M252F/T256D M252Y/T256QM252Y/P257L M252Y/P257N M252Y/V259I M252Y/V279Q M252Y/V308P/N434YM252Q/V308F M252Y/V308F

Table 4-2 is a continuation table of Table 4-1.

TABLE 4-2 M252Q/V308F/N434S M252Y/V308F/M428L M252Y/V308F/N434MM252Y/V308F/N434S M252Y/Y319I M252Q/M428L/N434S M252Y/M428L M252Y/N434MM252Y/N434S M252Y/N434A M252Y/N434Y S254T/V308F R255H/N434A R255Q/N434SR255H/N434S T256V/V308F T256P/Q311I T256P/I332E T256P/I332E/S440YT256P/E430Q T256P/N434H T256E/N434Y T256P/S440Y P257Y/V279Q P257L/V279EP257N/V279Q P257N/V279E P257N/V279Y P257L/V279Q P257N/{circumflex over( )}281S P257L/{circumflex over ( )}281S P257N/V284E P257N/L306YP257L/V308Y P257L/V308F P257N/V308Y P257I/Q311I/N434H P257L/Q311VP257L/G385N P257L/M428L P257I/E430Q P257I/N434H P257L/N434Y E258H/N434AE258H/N434H V259I/T307Q/V308F V259I/V308F V259I/V308F/Y319LV259I/V308F/Y319I V259A/V308F V259I/V308F/N434M V259I/V308F/N434SV259I/V308F/M428L/N434S V259I/V308F/M428L V259I/Y319I V259I/Y319I/N434SV259I/M428L V259I/M428L/N434S V259I/N434S

Table 4-3 is a continuation table of Table 4-2.

TABLE 4-3 V259I/N434Y V264I/A330L/I332E V264I/I332E D265F/N297E/I332ES267L/A327S E272R/V279L V279E/V284E V279Q/L306Y V279Y/V308F V279Q/V308FV279Q/G385H {circumflex over ( )}281S/V308Y {circumflex over( )}281S/V308F {circumflex over ( )}281S/N434Y E283F/V284E V284E/V308FV284E/G385H K288A/N434A K288D/H435K K288V/H435D T289H/N434A T289H/N434HL306I/V308F T307P/V308F T307Q/V308F/N434S T307Q/V308F/Y319L T307S/V308FT307Q/V308F T307A/E310A/N434A T307Q/E380A/N434A T307Q/M428L T307Q/N434MT307I/N434S T307V/N434S T307Q/N434S T307Q/N434Y V308T/L309P/Q311SV308F/L309Y V308F/Q311V V308F/Y319F V308F/Y319I/N434M V308F/Y319IV308F/Y319L V308F/Y319I/M428L V308F/Y319I/M428L/N434S V308F/Y319L/N434SV308F/I332E V308F/G385H V308F/M428L/N434M V308F/M428L V308F/M428L/N434SV308P/N434Y V308F/N434M V308F/N434S V308F/N434Y Q311G/N434S Q311D/N434SQ311E/N434S Q311N/N434S

Table 4-4 is a continuation table of Table 4-3.

TABLE 4-4 Q311Y/N434S Q311F/N434S Q311W/N434S Q311A/N434S Q311K/N434SQ311T/N434S Q311R/N434S Q311L/N434S Q311M/N434S Q311V/N434S Q311I/N434SQ311A/N434Y D312H/N434A D312H/N434H L314Q/N434S L314V/N434S L314M/N434SL314F/N434S L314I/N434S N315H/N434A N315H/N434H Y319I/V308F Y319I/M428LY319I/M428L/N434S Y319I/N434M Y319I/N434S L328H/I332E L328N/I332EL328E/I332E L328I/I332E L328Q/I332E L328D/I332E L328R/M428L/N434SA330L/I332E A330Y/I332E I332E/D376V I332E/N434S P343R/E345D D376V/E430QD376V/E430R D376V/N434H E380A/N434A G385R/Q386T/P387R/N389PG385D/Q386P/N389S N414F/Y416H M428L/N434M M428L/N434S M428L/N434AM428L/N434Y H429N/N434S E430D/N434S E430T/N434S E430S/N434S E430A/N434SE430F/N434S E430Q/N434S E430L/N434S E430I/N434S A431T/N434S

Table 4-5 is a continuation table of Table 4-4.

TABLE 4-5 A431S/N434S A431G/N434S A431V/N434S A431N/N434S A431F/N434SA431H/N434S L432F/N434S L432N/N434S L432Q/N434S L432H/N434S L432G/N434SL432I/N434S L432V/N434S L432A/N434S H433K/N434F H433L/N434S H433M/N434SH433A/N434S H433V/N434S H433K/N434S H433S/N434S H433P/N434S N434S/M428LN434S/Y436D N434S/Y436Q N434S/Y436M N434S/Y436G N434S/Y436E N434S/Y436FN434S/Y436T N434S/Y436R N434S/Y436S N434S/Y436H N434S/Y436K N434S/Y436LN434S/Y436V N434S/Y436W N434S/Y436I N434S/T437I

Herein, higher human FcRn-binding activity than that of native human IgGmeans that the human FcRn-binding activity is, for example, 105% ormore, preferably 110% or higher, 115% or higher, 120% or higher, 125% orhigher, particularly preferably 130% or higher, 135% or higher, 140% orhigher, 145% or higher, 150% or higher, 155% or higher, 160% or higher,165% or higher, 170% or higher, 175% or higher, 180% or higher, 185% orhigher, 190% or higher, 195% or higher, twice or higher, 2.5 fold orhigher, 3 fold or higher, 3.5 fold or higher, 4 fold or higher, 4.5 foldor higher, 5 fold or higher, 7.5 fold or higher, 10 fold or higher, 20fold or higher, 30 fold or higher, 40 fold or higher, 50 fold or higher,60 fold or higher, 70 fold or higher, 80 fold or higher, 90 fold orhigher, 100 fold or higher than that of native human IgG.

Such amino acid alterations can be appropriately introduced using knownmethods. For example, alterations in the Fc domain of human IgG1 aredescribed in Drug Metab Dispos. 2007 January 35(1): 86-94; Int Immunol.2006 Dec. 18, (12): 1759-69; J Biol. Chem. 2001 Mar. 2, 276(9):6591-604; J Biol. Chem. (2007) 282(3): 1709-17; J. Immunol. (2002)169(9): 5171-80; J. Immunol. (2009) 182(12): 7663-71; Molecular Cell,Vol. 7, 867-877, April, 2001; Nat. Biotechnol. 1997 Jul. 15, (7):637-40; Nat. Biotechnol. 2005 Oct. 23, (10): 1283-8; Proc Natl Acad SciUSA. 2006 Dec. 5, 103(49): 18709-14; EP 2154157; US 20070141052;WO2000/042072; WO2002/060919; WO2006/020114; WO2006/031370;WO2010/033279; WO2006/053301; and WO2009/086320.

According to Yeung et al. (The Journal of Immunology, 2009 182:7663-7671), the human FcRn-binding activity of human IgG1 is KD 1.7 μMunder an acidic pH range (pH6.0) condition but is almost undetectableunder a neutral pH range condition. Thus, preferred embodiments ofantigen-binding molecules provided by the present invention includeantigen-binding molecules of which human FcRn-binding activity under anacidic pH range condition is KD 20 μM or stronger and of which humanFcRn-binding activity under a neutral pH range condition is comparableto or higher than that of human IgG. More preferred embodiments includeantigen-binding molecules of which human FcRn-binding activity under anacidic pH range condition is KD 2.0 μM or stronger and of which humanFcRn-binding activity under a neutral pH range condition is KD 40 μM orstronger. Still more preferred embodiments include antigen-bindingmolecules of which human FcRn-binding activity under an acidic pH rangecondition is KD 0.5 μM or stronger and of which human FcRn-bindingactivity under a neutral pH range condition is KD 15 μM or stronger. Theabove KD values refers to values determined by the method described inThe Journal of Immunology, 2009 182: 7663-7671 (antigen-bindingmolecules are immobilized onto a chip, and human FcRn is injected as ananalyte).

In a preferred embodiment, the antigen-binding molecules provided by thepresent invention have human FcRn-binding activity at pH 7.0 and at 25°C. which is stronger than human IgG. In a more preferred embodiment,human FcRn-binding activity at pH 7.0 and at 25° C. is 28-fold strongerthan human IgG or stronger than KD 3.2 μM. In a more preferredembodiment, human FcRn-binding activity at pH 7.0 and at 25° C. is38-fold stronger than human IgG or stronger than KD 2.3 μM.

KD (dissociation constant) can be used as a value for human FcRn-bindingactivity. However, the human FcRn-binding activity of human IgG isalmost undetectable under a neutral pH range (pH 7.4) condition, and itis difficult to calculate the activity as KD. A method for assessingwhether the human FcRn-binding activity at pH 7.4 is higher than that ofhuman IgG is to assess based on the level of binding response in Biacorewhen analytes are injected at the same concentration. Specifically, ifthe response level when human FcRn is injected into a chip immobilizedwith an antigen-binding molecule provided by the present invention isgreater than the response level when human FcRn is injected into a humanIgG-immobilized chip, the human FcRn-binding activity of theantigen-binding molecule is concluded to be higher than that of thehuman IgG.

Fcγ receptor (FcγR) refers to a receptor capable of binding to the Fcregion of IgGs (for example, IgG1, IgG2, IgG3, or IgG4), and practicallyincludes any members belonging to the Fcγ receptor family. In human, thefamily includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb andFcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypeH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16) including isoform FcγRIIIa (includingallotype V158 and F158) and FcγRIIIb (including allotype FcγRIIIb-NA1and FcγRIIIb-NA2); as well as all unidentified human FcγRs, FcγRisoforms, and allotypes thereof. However, Fcγ receptor is not limited tothese examples. Without being limited thereto, origin of FcγR includeshumans, mice, rats, rabbits, and monkeys. FcγR may be derived from anyorganisms. Mouse FcγR includes, without being limited to, FcγRI (CD64),FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as wellas all unidentified mouse FcγRs, FcγR isoforms, and allotypes thereof.Such preferred Fcγ receptors include, for example, human FcγRI (CD64),FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI areshown in SEQ ID NOs: 39 (NM_(—)000566.3) and 40 (NP_(—)000557.1),respectively; the polynucleotide sequence and amino acid sequence ofhuman FcγRIIa (allotype H131) are shown in SEQ ID NOs: 41 (BC020823.1)and 42 (AAH20823.1) (allotype R131 is a sequence in which amino acid atposition 166 of SEQ ID NO: 42 is substituted with Arg), respectively;the polynucleotide sequence and amino acid sequence of FcγIIb are shownin SEQ ID NOs: 43 (BC146678.1) and 44 (AAI46679.1), respectively; thepolynucleotide sequence and amino acid sequence of FcγRIIIa are shown inSEQ ID NOs: 45 (BC033678.1) and 46 (AAH33678.1), respectively; and thepolynucleotide sequence and amino acid sequence of FcγRIIIb are shown inSEQ ID NOs: 47 (BC128562.1) and 48 (AAI28563.1), respectively (RefSeqaccession number is shown in each parentheses). Whether an Fcγ receptorhas binding activity to the Fc region of an IgG can be assessed by ALPHAscreen (Amplified Luminescent Proximity Homogeneous Assay), surfaceplasmon resonance (SPR)-based BIACORE method, and others (Proc. Natl.Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to FACS andELISA.

Meanwhile, “Fc ligand” or “effector ligand” refers to a molecule andpreferably a polypeptide that binds to an antibody Fc region, forming acomplex. The molecule may be derived from any organisms. The binding ofan Fc ligand to Fc region preferably induces one or more effectorfunctions. Such Fc ligands include, but are not limited to, Fcreceptors, FcγR, FcαR, FcεR, FcRn, C1q, and C3, mannan-binding lectin,mannose receptor, Staphylococcus Protein A, Staphylococcus Protein G,and viral FcγRs. The Fc ligands also include Fc receptor homologs (FcRH)(Davis et al., (2002) Immunological Reviews 190, 123-136), which are afamily of Fc receptors homologous to FcγR. The Fc ligands also includeunidentified molecules that bind to Fc.

FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16)including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is composedof two types of subunits, a chain that binds to the Fc region of IgG andcommon γ chain having ITAM responsible for transduction of intracellularactivation signal. Meanwhile, the cytoplasmic domain of FcγRII (CD32)including isoforms FcγRIIa (including allotypes H131 and R131) andFcγRIIc contains ITAM. These receptors are expressed on many immunecells such as macrophages, mast cells, and antigen-presenting cells. Theactivation signal transduced upon binding of these receptors to the Fcregion of IgG results in enhancement of the phagocytic activity andinflammatory cytokine production of macrophages, mast celldegranulation, and the activation of antigen-presenting cells. Fcγreceptors having the ability to transduce the activation signal asdescribed above are referred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1and FcγRIIb-2) contains ITIM responsible for transduction of inhibitorysignals. The crosslinking between FcγRIIb and B cell receptor (BCR) on Bcells suppresses the activation signal from BCR, which results insuppression of antibody production of B cells. The crosslinking ofFcγRIII and FcγRIIb on macrophages suppresses the phagocytic activityand inflammatory cytokine production. Fcγ receptors having the abilityto transduce the inhibitory signal as described above are referred to asinhibitory Fcγ receptors.

Receptor-binding domains that bind under a neutral pH range condition toFcγ receptors more strongly than native human IgG can be produced byaltering the amino acids of the Fc region of a human IgG. Suchalterations include, for example, substitution, insertion, and deletionof one or more amino acids. Alternatively, an antigen-binding domainthat binds to Fcγ receptor may be used as a receptor-binding domain.Such receptor-binding domains include Fab fragments that bind toFcγRIIIa, camel-derived single-domain antibodies, and the single-chainFvs, described in Protein Eng Des Sel. 2009 March; 22(3): 175-88;Protein Eng Des Sel. 2008 January; 21(1): 1-10; and J. Immunol. 2002Jul. 1; 169(1): 137-44; and FcγRI-binding cyclic peptides described inFASEB J. 2009 February; 23(2): 575-85. Whether the Fcγ receptor-bindingactivity of a receptor-binding domain is greater than that of the Fcregion of native human IgG can be appropriately assessed using themethods described above.

In the present invention, the activity of binding to a human Fcγreceptor under an acidic pH range condition means human Fcγreceptor-binding activity at pH 4.0 to pH 6.5, preferably human Fcγreceptor-binding activity at pH 5.0 to pH 6.5, more preferably human Fcγreceptor-binding activity at any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably human Fcγreceptor-binding activity at pH 5.8 to pH 6.0, which are close to the pHin the early endosome in vivo. Meanwhile, in the present invention, thebinding activity to a human Fcγ receptor under a neutral pH rangecondition means human Fcγ receptor-binding activity at pH 6.7 to pH10.0, preferably human Fcγ receptor-binding activity at pH 7.0 to pH9.0, more preferably human Fcγ receptor-binding activity at any of pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, andparticularly preferably human Fcγ receptor-binding activity at pH 7.4,which is close to the pH of plasma in vivo.

Regarding measurement temperature, the binding affinity between areceptor-binding domain and a human Fcγ receptor may be measured at anytemperature between 10° C. and 50° C. The binding affinity between areceptor-binding domain and a human Fcγ receptor is preferablydetermined at 15° C. to 40° C., more preferably at any of 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35° C. Without beingparticularly limited, the 25° C. temperature is a preferred embodiment.

Receptor-binding domains of the present invention preferably include,for example, the Fc regions of human IgGs. The origin of such an Fcregion is not particularly limited, and the domain can be obtained fromany nonhuman animals or from humans. Nonhuman animals preferably includemice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats,sheep, bovines, horses, camels, and nonhuman primates. In anotherembodiment, the receptor-binding domains can be obtained from cynomolgusmonkeys, marmosets, rhesus monkeys, chimpanzees, and humans. The Fcregions are preferably obtained from the Fc region of human IgG1, andare not limited to particular IgG classes. Specifically, the Fc regionof human IgG1, IgG2, IgG3, or IgG4 can be suitably used as areceptor-binding domain. Naturally occurring or artificially modifiedIgG variants include, for example, those described in publisheddocuments (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91; Curr. Opin.Immunol. (2008) 20 (4), 460-470; Protein Eng. Des. Sel. (2010) 23 (4),195-202; WO2009/086320; WO2008/092117; WO2007/041635; and WO2006/105338)but are not limited thereto.

As long as the receptor-binding domain binds under a neutral pH rangecondition to Fcγ receptor more strongly than native human IgG, aminoacids may be altered at any positions. When receptor-binding domains areproduced by altering the Fc region of human IgG1, amino acid alterationsfor increasing the binding activity to an Fcγ receptor under a neutralpH range condition includes, for example, the amino acid alterationsdescribed in WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072,WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635,WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260, andWO2006/023403.

Preferred amino acids when modifying the Fc region of IgG forreceptor-binding domains that bind to human Fcγ receptor are, forexample, at least one or more amino acids selected from the groupconsisting of the amino acids at positions 221, 222, 223, 224, 225, 227,228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243,244, 245, 246, 247, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292,293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307,308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339,341, 343, 375, 376, 377, 378, 379, 380, 382, 385, 386, 387, 389, 392,396, 421, 423, 427, 428, 429, 430, 431, 433, 434, 436, 438, 440, and 442according to EU numbering. Alteration of these amino acids increases theactivity of an IgG Fc region to bind an Fcγ receptor under a neutral pHrange condition.

Particularly preferred alterations for increasing the binding to an Fcγreceptor under a neutral pH range condition include, for example,alteration of at least one or more amino acids selected from the groupconsisting of:

the amino acid at position 221 which is Lys or Tyr;the amino acid at position 222 which is Phe, Trp, Glu, or Tyr;the amino acid at position 223 which is Phe, Trp, Glu, or Lys;the amino acid at position 224 which is Phe, Trp, Glu, or Tyr;the amino acid at position 225 which is Glu, Lys, or Trp;the amino acid at position 227 which is Glu, Gly, Lys, or Tyr;the amino acid at position 228 which is Glu, Gly, Lys, or Tyr;the amino acid at position 230 which is Ala, Glu, Gly, or Tyr;the amino acid at position 231 which is Glu, Gly, Lys, Pro, or Tyr;the amino acid at position 232 which is Glu, Gly, Lys, or Tyr;the amino acid at position 233 which is Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 234 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 235 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 236 which is Ala, Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 237 which is Ala, Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 238 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 239 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;the amino acid at position 240 which is Ala, Ile, Met, or Thr;the amino acid at position 241 which is Asp, Glu, Leu, Arg, Trp, or Tyr;the amino acid at position 243 which is Leu, Glu, Leu, Gln, Arg, Trp, orTyr;the amino acid at position 244 which is His;the amino acid at position 245 which is Ala;the amino acid at position 246 which is Asp, Glu, His, or Tyr;the amino acid at position 247 which is Ala, Phe, Gly, His, Ile, Leu,Met, Thr, Val, or Tyr;the amino acid at position 249 which is Glu, His, Gln, or Tyr;the amino acid at position 250 which is Glu or Gln;the amino acid at position 251 which is Phe;the amino acid at position 254 which is Phe, Met, or Tyr;the amino acid at position 255 which is Glu, Leu, or Tyr;the amino acid at position 256 which is Ala, Met, or Pro;the amino acid at position 258 which is Asp, Glu, His, Ser, or Tyr;the amino acid at position 260 which is Asp, Glu, His, or Tyr;the amino acid at position 262 which is Ala, Glu, Phe, Ile, or Thr;the amino acid at position 263 which is Ala, Ile, Met, or Thr;the amino acid at position 264 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 265 which is Ala, Glu, Leu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 266 which is Ala, Phe, Ile, Leu, Met, or Thr;the amino acid at position 267 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;the amino acid at position 268 which is Ala, Asp, Glu, Phe, Gly, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Trp;the amino acid at position 269 which is Asp, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 270 which is Glu, Phe, Gly, His, Ile, Leu,Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 271 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 272 which is Asp, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 273 which is Phe or Ile;the amino acid at position 274 which is Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 275 which is Leu or Trp;the amino acid at position 276 which is Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 278 which is Asp, Glu, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;the amino acid at position 279 which is Ala;the amino acid at position 280 which is Ala, Gly, His, Lys, Leu, Pro,Gln, Trp, or Tyr;the amino acid at position 281 which is Asp, Lys, Pro, or Tyr;the amino acid at position 282 which is Glu, Gly, Lys, Pro, or Tyr;the amino acid at position 283 which is Ala, Gly, His, Ile, Lys, Leu,Met, Pro, Arg, or Tyr;the amino acid at position 284 which is Asp, Glu, Leu, Asn, Thr, or Tyr;the amino acid at position 285 which is Asp, Glu, Lys, Gln, Trp, or Tyr;the amino acid at position 286 which is Glu, Gly, Pro, or Tyr;the amino acid at position 288 which is Asn, Asp, Glu, or Tyr;the amino acid at position 290 which is Asp, Gly, His, Leu, Asn, Ser,Thr, Trp, or Tyr;the amino acid at position 291 which is Asp, Glu, Gly, His, Ile, Gln, orThr;the amino acid at position 292 which is Ala, Asp, Glu, Pro, Thr, or Tyr;the amino acid at position 293 which is Phe, Gly, His, Ile, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 294 which is Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 295 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 296 which is Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val;the amino acid at position 297 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 298 which is Ala, Asp, Glu, Phe, His, Ile,Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr;the amino acid at position 299 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;the amino acid at position 300 which is Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;the amino acid at position 301 which is Asp, Glu, His, or Tyr;the amino acid at position 302 which is Ile;the amino acid at position 303 which is Asp, Gly, or Tyr;the amino acid at position 304 which is Asp, His, Leu, Asn, or Thr;the amino acid at position 305 which is Glu, Ile, Thr, or Tyr;the amino acid at position 311 which is Ala, Asp, Asn, Thr, Val, or Tyr;the amino acid at position 313 which is Phe;the amino acid at position 315 which is Leu;the amino acid at position 317 which is Glu or Gln;the amino acid at position 318 which is His, Leu, Asn, Pro, Gln, Arg,Thr, Val, or Tyr,the amino acid at position 320 which is Asp, Phe, Gly, His, Ile, Leu,Asn, Pro, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 322 which is Ala, Asp, Phe, Gly, His, Ile,Pro, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 323 which is Ile, Leu, or Met;the amino acid at position 324 which is Asp, Phe, Gly, His, Ile, Leu,Met, Pro, Arg, Thr, Val, Trp, or Tyr;the amino acid at position 325 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 326 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 327 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr;the amino acid at position 328 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 329 which is Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 330 which is Cys, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 331 which is Asp, Phe, His, Ile, Leu, Met,Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 332 which is Ala, Asp, Glu, Phe, Gly, His,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 333 which is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, or Tyr;the amino acid at position 334 which is Ala, Glu, Phe, His, Ile, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 335 which is Asp, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr;the amino acid at position 336 which is Glu, Lys, or Tyr;the amino acid at position 337 which is Asp, Glu, His, or Asn;the amino acid at position 339 which is Asp, Phe, Gly, Ile, Lys, Met,Asn, Gln, Arg, Ser, or Thr;the amino acid at position 376 which is Ala or Val;the amino acid at position 377 which is Gly or Lys;the amino acid at position 378 which is Asp;the amino acid at position 379 which is Asn;the amino acid at position 380 which is Ala, Asn, or Ser;the amino acid at position 382 which is Ala or Ile;the amino acid at position 385 which is Glu;the amino acid at position 392 which is Thr;the amino acid at position 396 which is Asp, Glu, Phe, Ile, Lys, Leu,Met, Gln, Arg, or Tyr;the amino acid at position 421 which is Lys;the amino acid at position 427 which is Asn;the amino acid at position 428 which is Phe or Leu;the amino acid at position 429 which is Met;the amino acid at position 434 which is Trp;the amino acid at position 436 which is Ile; andthe amino acid at position 440 is Gly, His, Ile, Leu, or Tyr, accordingto EU numbering in the Fc region. Further, the number of amino acids tobe altered is not particularly limited, and amino acids may be modifiedat only one site, or two or more sites. Combinations of amino acidalterations at two or more sites include, for example, those describedin Tables 5-1 to 5-3.

TABLE 5-1 AMINO ACID COMBINATION AMINO ACID COMBINATIONK370E/P396L/D270E S239Q/I332Q Q419H/P396L/D270E S267D/I332EV240A/P396L/D270E S267E/I332E R255L/P396L/D270E S267L/A327SR255L/P396L/D270E S267Q/A327S R255L/P396L/D270E/R292G S298A/I332ER255L/P396L/D270E S304T/I332E R255L/P396L/D270E/Y300L S324G/I332DF243L/D270E/K392N/P396L S324G/I332E F243L/R255L/D270E/P396L S324I/I332DF243L/R292P/Y300L/V305I/P396L S324I/I332E F243L/R292P/Y300L/P396LT260H/I332E F243L/R292P/Y300L T335D/I332E F243L/R292P/P396L V240I/V266IF243L/R292P/V305I V264I/I332E F243L/R292P D265F/N297E/I332ES298A/E333A/K334A D265Y/N297D/I332E E380A/T307A F243L/V262I/V264WK326M/E333S N297D/A330Y/I332E K326A/E333A N297D/T299E/I332E S317A/K353AN297D/T299F/I332E A327D/I332E N297D/T299H/I332E A330L/I332EN297D/T299I/I332E A330Y/I332E N297D/T299L/I332E E258H/I332EN297D/T299V/I332E E272H/I332E P230A/E233D/I332E E272I/N276DP244H/P245A/P247V E272R/I332E S239D/A330L/I332E E283H/I332ES239D/A330Y/I332E E293R/I332E S239D/H268E/A330Y F241L/V262IS239D/I332E/A327A P241W/F243W S239D/I332E/A330I

Table 5-2 is a continuation of Table 5-1.

TABLE 5-2 F243L/V264I S239D/N297D/I332E H268D/A330Y S239D/S298A/I332EH268E/A330Y S239D/V264I/I332E K246H/I332E S239E/N297D/I332E L234D/I332ES239E/V264I/I332E L234E/I332E S239N/A330L/I332E L234G/I332ES239N/A330Y/I332E L234I/I332E S239N/S298A/I332E L234I/L235DS239Q/V264I/I332E L234Y/I332E V264E/N297D/I332E L235D/I332EV264I/A330L/T332E L235E/I332E V264I/A330Y/I332E L235I/I332EV264T/S298A/I332E L235S/I332E Y296D/N297D/I332E L328A/I332DY296E/N297D/I332E L328D/I332D Y296H/N297D/I332E L328D/I332EY296N/N297D/I332E L328E/I332D Y296Q/N297D/I332E L328E/I332EY296T/N297D/I332E L323F/I332D D265Y/N297D/T299L/I332E L328F/I332EF241E/F243Q/V262T/V264E L328H/I332E F241E/F243R/V262E/V264R L328I/I332DF241E/F243Y/V262T/V264R L328I/I332E F241L/F243L/V262I/V264I L328M/I332DF241R/F243Q/V262T/V264R L328M/I332E F241S/F243H/V262T/V264T L328N/I332DF241W/F243W/V262A/V264A L328N/I332E F241Y/F243Y/V262T/V264T L328Q/I332DI332E/A330Y/H268E/A327A L328Q/I332E N297D/I332E/S239D/A330L L328T/I332DN297D/S298A/A330Y/I332E L328T/I332E S239D/A330Y/I332E/K326E L328V/I332DS239D/A330Y/I332E/K326T L328V/I332E S239D/A330Y/I332E/L234I L328Y/I332DS239D/A330Y/I332E/L235D

Table 5-3 is a continuation of Table 5-2.

TABLE 5-3 L328Y/I332E S239D/A330Y/I332E/V240I N297D/I332ES239D/A330Y/I332E/V264T N297E/I332E S239D/A330Y/I332E/V266I N297S/I332ES239D/D265F/N297D/I332E P227G/I332E S239D/D265H/N297D/I332E P230A/E233DS239D/D265I/N297D/I332E Q295E/I332E S239D/D265L/N297D/I332E R255Y/I332ES239D/D265T/N297D/I332E S239D/I332D S239D/D265V/N297D/I332E S239D/I332ES239D/D265Y/N297D/I332E S239D/I332N S239D/I332E/A330Y/A327A S239D/I332QS239D/I332E/H268E/A327A S239E/D265G S239D/I332E/H268E/A330Y S239E/D265NS239D/N297D/I332E/A330Y S239E/D26SQ S239D/N297D/I332E/K326E S239E/I332DS239D/N297D/I332E/L235D S239E/I332E S239D/V264I/A330L/I332E S239E/I332NS239D/V264I/S298A/I332E S239E/I332Q S239E/V264I/A330Y/I332E S239N/I332DF241E/F243Q/V262T/V264E/I332E S239N/I332E F241E/F243R/V262E/V264R/I332ES239N/I332N F241E/F243Y/V262T/V264R/I332E S239N/I332QP241R/F243Q/V262T/V264R/I332E S239Q/I332D S239D/I332E/H268E/A330Y/A327AS239Q/I332E S239E/V264I/S298A/A330Y/I332E S239Q/I332NF241Y/F243Y/V262T/V264T/N297D/I332E S267E/L328F G236D/S267E S239D/S267E

Herein, the activity to bind a human Fcγ receptor is deemed to begreater than that of native human IgG when the activity to bind humanFcγ receptors FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and/or FcγRIIIb, isgreater than that of native human IgG. This means that, for example, theactivity to bind a human Fcγ receptor is 105% or more, preferably 110%or more, 115% or more, 120% or more, 125% or more, particularlypreferably 130% or more, 135% or more, 140% or more, 145% or more, 150%or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% ormore, 180% or more, 185% or more, 190% or more, 195% or more, twice ormore, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times ormore, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times ormore, 20 times or more, 30 times or more, 40 times or more, 50 times ormore, 60 times or more, 70 times or more, 80 times or more, 90 times ormore, or 100 times or more than that of native human IgG.

Receptor-binding domains of the present invention may have the propertyof having a greater binding activity to a specific Fcγ receptor(s) thanthe binding activity to other Fcγ receptors (selectively binds to aspecific Fcγ receptor(s)). Examples include a receptor-binding domainhaving a greater binding activity to an inhibitory Fcγ receptor than toan activating Fcγ receptor. Such receptor-binding domains preferablyinclude those having a greater binding activity to FcγRIIb (includingFcγRIIb-1 and FcγRIIb-2), which is an inhibitory Fcγ receptor, than toan activating Fcγ receptor selected from:

FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 andF158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2);andFcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 andR131) and FcγRIIc. The receptor-binding domains particularly preferablyinclude those having a greater binding activity to FcγRIIb-1 and/orFcγRIIb-2 than to FcγRIIa (allotype H131).

Whether a receptor-binding domain has the property of selectivelybinding to a specific Fcγ receptor can be determined by measuring andcomparing the KD value of the receptor-binding domain for each Fcγreceptor. For example, when the KD value of a receptor-binding domain toan activating Fcγ receptor divided by its KD value to an inhibitory Fcγreceptor is 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 ormore, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 ormore, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 ormore, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 110or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 ormore, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more,220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 ormore, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more,330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 ormore, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more,440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 ormore, 500 or more, 520 or more, 540 or more, 560 or more, 580 or more,600 or more, 620 or more, 640 or more, 660 or more, 680 or more, 700 ormore, 720 or more, 740 or more, 760 or more, 780 or more, 800 or more,820 or more, 840 or more, 860 or more, 880 or more, 900 or more, 920 ormore, 940 or more, 960 or more, 980 or more, 1000 or more, 1500 or more,2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more,4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more,7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more,9500 or more, 10000 or more, or 100000 or more, it is determined thatthe receptor-binding domain may bind more selectively to the inhibitoryFcγ receptor than to the activating Fcγ receptor.

Without being particularly limited thereto, receptor-binding domainshaving a greater binding activity to an inhibitory Fcγ receptor than toan activating Fcγ receptor (selectively binds to an inhibitory Fcγreceptor) preferably include, for example, the IgG Fc region variantsdescribed in WO2012/115241, such as Fc regions with alterations of theamino acids at positions 238 and/or 328 (EU numbering) into differentamino acids in IgG Fc region, more preferably Fc regions with alterationof the amino acid at position 238 into Asp and/or alteration of theamino acid at position 328 into Glu. Furthermore, it is possible toselect appropriate IgG Fc region variants described in US2009/0136485.

At least one different alteration may be added to IgG Fc regions incombination with the above-described alterations. It is preferable thatas a result of the alteration, the binding activity to FcγRIIb isincreased, and the binding activity to FcγRIIa (allotype H131) andFcγRIIa (allotype R131) is maintained or is reduced. Such alterationimproves the binding selectivity for FcγRIIb over FcγRIIa. Alterationthat improves the binding selectivity for FcγRIIb over FcγRIIa (allotypeR131) is preferred, and alteration that improves the binding selectivityfor FcγRIIb over FcγRIIa (allotype R131) and FcγRIIa (allotype H131) ismore preferred. Without being particularly limited thereto, suchalterations include, for example, at least one or more amino acidalterations selected from the following group:

the amino acid at position 233 is Asp;the amino acid at position 234 is Trp or Tyr;the amino acid at position 235 is Phe, Trp, or Tyr;the amino acid at position 236 is Asp,the amino acid at position 237 is Ala, Asp, Glu, Phe, Leu, Met, Trp, orTyr;the amino acid at position 238 is Phe or Leu;the amino acid at position 239 is Asp, Glu, Gly, Leu, or Asn;the amino acid at position 266 is Ile, Leu, or Met;the amino acid at position 267 is Ala, Asp, Glu, Ile, Met, Gln, or Val;the amino acid at position 268 is Ala, Asp, Glu, Gly, Asn, or Gln;the amino acid at position 271 is Gly or Leu;the amino acid at position 295 is Leu;the amino acid at position 296 is Asp;the amino acid at position 300 is Asp, Glu, or Gln;the amino acid at position 323 is Ile, Leu, or Met;the amino acid at position 324 is Ile or Val;the amino acid at position 325 is Met or Ser;the amino acid at position 326 is Ala, Asp, Glu, Phe, His, Ile, Leu,Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 327 is Asp, Glu, Gly, or Asn;the amino acid at position 328 is Ala, Asp, Phe, Ile, Met, Gln, Ser,Thr, Val, Trp, or Tyr;the amino acid at position 330 is Lys, Met, or Arg;the amino acid at position 331 is Phe, Trp, or Tyr;the amino acid at position 332 is Phe;the amino acid at position 333 is Pro;the amino acid at position 334 is Ala, Trp, Glu, Phe, His, Ile, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr;the amino acid at position 335 is Asp; andthe amino acid at position 337 is Asp according to EU numbering.

Among the above, preferred alterations include, for example, at leastone or more amino acid alterations selected from the following group:

the amino acid at position 233 is Asp;the amino acid at position 234 is Trp or Tyr;the amino acid at position 237 is Ala, Asp, Glu, Phe, Leu, Met, Trp, orTyr;the amino acid at position 239 is Asp;the amino acid at position 267 is Ala, Gln, or Val;the amino acid at position 268 is Asp, Glu, or Asn;the amino acid at position 271 is Gly;the amino acid at position 296 is Asp;the amino acid at position 323 is Ile, Leu, or Met;the amino acid at position 326 is Ala, Asp, Glu, Leu, Met, Asn, Gln,Ser, or Thr; andthe amino acid at position 330 is Lys, Met, or Arg according to EUnumbering.

The alterations described above may be an alteration at one site, or attwo or more sites in combination. Preferred alterations include, forexample, the alterations shown in Tables 24 to 25, 27 to 34, and 36 to37.

Without being particularly limited thereto, one embodiment of thereceptor-binding domain included in antigen-binding molecules of thepresent invention includes altered Fc regions of human IgG1 (SEQ ID NO:49), IgG2 (SEQ ID NO: 50), IgG3 (SEQ ID NO: 51), and IgG4 (SEQ ID NO:52). Examples of the altered Fc regions include the Fc regions of humanIgGs (IgG1, IgG2, IgG3, and IgG4), in which the amino acid at position238 (EU numbering) is Asp and the amino acid at position 271 (EUnumbering) is Gly. The Fc regions of human IgGs (IgG1, IgG2, IgG3, andIgG4) in which the amino acid at position 238 (EU numbering) is Asp andthe amino acid at position 271 is Gly, and antigen-binding moleculescontaining the Fc regions exhibit a greater binding activity to theinhibitory Fcγ receptor rather than to the activating Fcγ receptor.

In the present invention, at least one different alteration may be addedto the Fc region in which the amino acid at position 238 (EU numbering)is Asp and the amino acid at position 271 is Gly (EU numbering). It ispreferable that as a result, the binding activity to FcγRIIb-1 and/orFcγRIIb-2 is increased, and the binding activity to FcγRIIa (allotypeH131) and FcγRIIa (allotype R131) is maintained or is reduced. It isalso preferable that the level of increase in the binding activity tothe inhibitory Fcγ receptor (FcγRIIb-1 and/or FcγRIIb-2) is higher thanthe level of increase in the binding activity to the activating Fcγreceptor (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa (allotype V158), FcγRIIIa(allotype F158), FcγRIIIb (allotype FcγRIIIb-NA1), FcγRIIIb (allotypeFcγRIIIb-NA2), FcγRIIa (allotype H131), and FcγRIIa (allotype R131)).Such alteration improves the binding selectivity for FcγRIIb overFcγRIIa.

Without being particularly limited thereto, selective receptor-bindingdomains include, for example, Fc regions of a human IgG (IgG1, IgG2,IgG3, or IgG4) in which the amino acid at position 238 (EU numbering) isaltered to Asp and the amino acid at position 271 is altered to Gly, andin which one or more of the amino acids at positions 233, 234, 237, 244,245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 264, 265,266, 267, 268, 269, 270, 272, 279, 283, 285, 286, 288, 293, 296, 307,308, 309, 311, 312, 314, 316, 317, 318, 326, 327, 330, 331, 332, 333,339, 341, 343, 375, 376, 377, 378, 380, 382, 385, 386, 387, 389, 396,423, 427, 428, 430, 431, 433, 434, 436, 438, 440, and 442 according toEU numbering are altered.

Furthermore, without being particularly limited thereto, selectivereceptor-binding domains include, for example, Fc regions of a human IgG(IgG1, IgG2, IgG3, or IgG4) in which the amino acid at position 238 (EUnumbering) is altered to Asp and the amino acid at position 271 isaltered to Gly, and in which one or more amino acid alterations selectedfrom the following group are made:

the amino acid at position 233 is Asp;the amino acid at position 234 is Tyr;the amino acid at position 237 is Asp;the amino acid at position 264 is Ile;the amino acid at position 265 is Glu;the amino acid at position 266 is Phe, Met, or Leu;the amino acid at position 267 is Ala, Glu, Gly, or Gln;the amino acid at position 268 is Asp or Glu;the amino acid at position 269 is Asp;the amino acid at position 272 is Asp, Phe, Ile, Met, Asn, or Gln;the amino acid at position 296 is Asp;the amino acid at position 326 is Ala or Asp;the amino acid at position 327 is Gly;the amino acid at position 330 is Lys or Arg;the amino acid at position 331 is Ser;the amino acid at position 332 is Thr;the amino acid at position 333 is Thr, Lys, or Arg; andthe amino acid at position 396 is Asp, Glu, Phe, Ile, Lys, Leu, Met,Gln, Arg, or Tyr, according to EU numbering.

Without being particularly limited thereto, one embodiment of theabove-described Fc regions includes, for example, those described inTables 6-1 to 6-6.

TABLE 6-1 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP208E233D/G237D/P238D/H268D/P271G/A330R BP209G237D/P238D/H268D/P271G/K326A/A330R BP210 G237D/P238D/H268D/P271G/A330RBP211 E233D/P238D/H268D/P271G/K326A/A330R BP212E233D/P238D/H268D/P271G/Y296D/A330R BP213 E233D/P238D/H268D/P271G/A330RBP214 E233D/L234Y/G237D/P238D/Y296D/K326D/A330K BP215G237D/P238D/H268D/P271G/Y296D/A330K BP216G237D/P238D/S2670/H268D/P271G/A330K BP217G237D/P238D/S267Q/H268D/P271G/Y296D/A330K BP218G237D/P238D/H268D/P271G/K326D/A330K BP219L234Y/G237D/P238D/H268D/P271G/A330K BP220E233D/G237D/P238D/H268D/P271G/Y296D/A330K BP221L234Y/G237D/P238D/Y296D/K326A/A330K BP222L234Y/G237D/P238D/P271G/K326A/A330R BP223L234Y/G237D/P238D/H268D/P271G/K326A/A330R BP224L234Y/G237D/P238D/S267Q/H268D/P271G/K326A/A330R BP225L234Y/G237D/P238D/K326D/A330R BP226 L234Y/G237D/P238D/P271G/K326D/A330RBP227 L234Y/G237D/P238D/H268D/P271G/K326D/A330R BP228L234Y/G237D/P238D/S267Q/H268D/P271G/K326D/A330R BP229E233D/L234Y/G237D/P238D/P271G/K326A/A330R BP230E233D/G237D/P238D/H268D/P271G/Y296D/A330R BP231G237D/P238D/H268D/P271G/Y296D/A330R BP232L234Y/G237D/P238D/P271G/K326A/A330K BP233 L234Y/G237D/P238D/P271G/A330KBP234 E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330K BP235E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/K326D/A330R BP236E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330R BP237E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330K

Table 6-2 is a continuation table of Table 6-1.

TABLE 6-2 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP238E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/K326A/A330R BP239E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330R BP240E233D/G237D/P238D/S267Q/H268D/P271G/A330R BP241E233D/G237D/P238D/H268D/P271G/K326D/A330R BP242E233D/G237D/P238D/M268D/P271G/K326A/A330R BP243E233D/L234Y/G237D/P238D/H268D/P271G/A330R BP244E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/A330R BP245E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330R BP246E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330R BP247E233D/G237D/P238D/H268D/P271G/Y296D/K326D/A330R BP248E233D/G237D/P238D/H268D/P271G/Y296D/K326A/A330R BP249E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/A330R BP262G237D/P238D/H268E/P271G BP264 E233D/G237D/P238D/H268E/P271G/Y296D/A330RBP265 G237D/P238D/H268E/P271G/Y296D/A330R BP266E233D/G237D/P238D/H268E/P271G/A330R BP267 E233D/G237D/P238D/H268E/P271GBP268 E233D/G237D/P23SD/H268E/P271G/Y296D BP269G237D/P238D/H268E/P271G/Y296D BP300 E233D/G237D/P238D/V264I/H268E/P271GBP313 E233D/G237D/P238D/D265E/H268E/P271G BP333E233D/G237D/P238D/V266F/H268E/P271G BP338E233D/G237D/P238D/V266L/H268E/P271G BP339E233D/G237D/P238D/V266M/H268E/P271G BP348E233D/G237D/P238D/S267A/H268E/P271G BP350E233D/G237D/P238D/S267E/H268E/P271G BP352E233D/G237D/P238D/S267G/H268E/P271G BP367E233D/G237D/P238D/H268E/E269D/P271G BP384E233D/G237D/P238D/H268D/P271G/Y296D/A330R/K334R BP390E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332S BP391E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332T

Table 6-3 is a continuation table of Table 6-2.

TABLE 6-3 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP392E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/I332K BP393E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/I332R BP423E233D/G237D/P238D/S267A/H268E/P271G/ A330R BP425E233D/G237D/P238D/V266L/S267A/H268E/ P271G/A330R BP426E233D/G237D/P238D/S267A/H268E/E269D/ P271G/A330R BP427E233D/G237D/P238D/S267A/H268E/E269Y/ P271G/A330R BP428E233D/G237D/P238D/S267G/H268E/P271G/ A330R BP429E233D/G237D/P238D/V264I/S267G/H268E/ P271G/A330R BP430E233D/G237D/P238D/V266L/S267G/H268E/ P271G/A330R BP431E233D/G237D/P238D/S267G/H268E/E269D/ P271G/A330R BP432E233D/G237D/P238D/S267G/H268E/E269Y/ P271G/A330R BP433E233D/G237D/P238D/H268D/P271G/Y296D/ A330K/I332T BP434E233D/G237D/P238D/H268D/P271G/Y296D/ K326D/A330R/I332T BP435E233D/G237D/P238D/H268D/P271G/Y296D/ K326A/A330R/I332T BP436E233D/G237D/P238D/S267A/H268E/P271G/ Y296D/A330R/I332T BP437G237D/P238D/S267A/H268E/P271G/Y296D/ A330R/I332T BP438E233D/G237D/P238D/S267A/H268E/P271G/ A330R/I332T BP439E233D/G237D/P238D/V264I/V266L/S267A/ H268E/P271G/A330R BP440E233D/G237D/P238D/V264I/H268E/P271G/ A330R BP441E233D/G237D/P238D/V266L/H268E/P271G/ A330R BP442E233D/G237D/P238D/H268E/E269D/P271G/ A330R BP443E233D/G237D/P238D/V266L/H268E/E269D/ P271G/A330R BP444E233D/G237D/P238D/H268E/E269N/P271G/ A330R BP445E233D/G237D/P238D/V264I/S267A/H268E/ P271G/A330R BP446E233D/G237D/P238D/S267A/H268E/E269N/ P271G/A330R BP447E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396A BP448E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396D BP449E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396E BP450E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396F BP451E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396G BP452E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396H

Table 6-4 is a continuation table of Table 6-3.

TABLE 6-4 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP453E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396I BP454E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396K BP455E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396L BP456E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396M BP457E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396N BP458E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396Q BP459E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396R BP460E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396S BP461E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396T BP462E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396V BP463E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396W BP464E233D/G237D/P238D/S267A/H268E/P271G/ A330R/P396Y BP465E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E333K BP466E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E333R BP467E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E334S BP468E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E334T BP469E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E333S BP470E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/E333T BP471E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/P331S BP472E233D/G237D/P238D/H268D/P271G/Y296D/ A330S BP473E233D/G237D/P238D/H268D/P271G/Y296D/ A327G/A330R BP474E233D/G237D/P238D/H268D/P271G/Y296D/ A330R/P331S BP475E233D/G237D/P238D/H268D/P271G/Y296D/ A327G/A330S BP476E233D/G237D/P238D/H268D/P271G/Y296D/ A327G/A330S/P331S BP477E233D/G237D/P238D/H268D/P271G/Y296D/ A327G/A330R/P331S BP478E233D/G237D/P238D/H268D/P271G/Y296D/ A330R +S131C/K133R/G137E/G138S/Q196K/ I199T/N203D/K214R/P217S + 219-221DELETION + K222Y/T223G/H224P/T225P BP479E233D/G237D/P238D/V264I/V266L/S267A/ H268E/P271G BP480E233D/G237D/P238D/V266L/H268E/E269D/ P271G BP481E233D/G237D/P238D/V264I/S267A/H268E/ P271G

Table 6-5 is a continuation table of Table 6-4.

TABLE 6-5 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP482E233D/G237D/P238D/S267A/H268E/ E269N/P271G BP483E233D/G237D/P238D/V266L/S267A/ H268E/P271G BP484E233D/G237D/P238D/S267A/H268E/ E269D/P271G BP485E233D/G237D/P238D/S267A/H268E/ E269Y/P271G BP487E233D/G237D/P238D/V264I/S267A/ H268E/P271G/A330R/P396M BP488E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D/A330R BP489E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D/A330R/P396M BP490G237D/P238D/V264I/S267A/H268E/ P271G/A330R BP491G237D/P238D/V264I/S267A/H268E/ P271G/Y296D/A330R BP492P238D/V264I/S267A/H268E/P271G BP493 P238D/V264I/S267A/H268E/P271G/ Y296DBP494 G237D/P238D/S267A/H268E/P271G/ Y296D/A330R BP495G237D/P238D/S267G/H268E/P271G/ Y296D/A330R BP496E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D BP497E233D/G237D/P238D/V264I/S267A/ H268E/P271G/A327G/A330R BP498E233D/G237D/P238D/V264I/S267A/ H268E/P271G/A330R/P396L BP499E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D/A330R/P396L BP500G237D/P238D/V264I/S267A/H268E/ P271G/Y296D BP501G237D/P238D/V264I/S267A/H268E/ P271G BP502E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D/A327G/A330R BP503E233D/G237D/P238D/V264I/S267A/ H268E/P271G/Y296D/A327G/A330R/P396M BP504E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272P BP505E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272D BP506E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272P/Y296D/A330R BP507E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272P/A330R BP508E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272P/Y296D BP509E233D/G237D/P238D/V264I/S267A/ H268E/P271G/E272D/Y296D BP510G237D/P238D/V264I/S267A/H268E/ P271G/E272P/A330R BP511G237D/P238D/V264I/S267A/H268E/ P271G/E272P/Y296D/A330R BP513E233D/G237D/P238D/H268E/E272D/ P271G

Table 6-6 is a continuation table of Table 6-5.

TABLE 6-6 ALTERED Fc REGION ALTERED AMINO ACIDS (EU NUMBERING) BP514E233D/G237D/P238D/H268E/E272F/P271G BP517E233D/G237D/P238D/H268E/E272I/P271G BP520E233D/G237D/P238D/H268E/E272M/P271G BP521E233D/G237D/P238D/H268E/E272N/P271G BP523E233D/G237D/P238D/H268E/E272Q/P271G

It is preferable that the above-described receptor-binding domains whichbind to human Fcγ receptor further contain amino acid alterations thatenhance the FcRn-binding under an acidic pH range condition. Amino acidsthat can be altered as such include, for example:

the amino acids at positions 252, 254, 256, 309, 311, 315, 433, and/or434 (EU numbering), and in combination with these described above, theamino acids at positions 253, 310, 435, and/or 426 (EU numbering), asdescribed in WO1997/034631;the amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272,286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376,378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439,and/or 447 (EU numbering), as described in WO2000/042072; the aminoacids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385,386, 387, 389, 428, 433, 434, and/or 436 (EU numbering), as described inWO2002/060919;the amino acids at positions 250, 314, and 428 (EU numbering), asdescribed in WO2004/092219;the amino acids at positions 238, 244, 245, 249, 252, 256, 257, 258,260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316,317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427,430, 431, 434, 436, 438, 440, and/or 442 (EU numbering), as described inWO2006/020114; andthe amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434,and/or 436 (EU numbering), as described in WO2010/045193. Alterations ofthese amino acids enhance the FcRn-binding of IgG Fc regions under anacidic pH range condition.

More specifically, such alterations include, for example, at least oneor more amino acid alterations selected from the following group:

the amino acid at position 244 is Leu;the amino acid at position 245 is Arg;the amino acid at position 249 is Pro;the amino acid at position 250 is Gln or Glu;the amino acid at position 251 is Arg, Asp, Glu, or Leu;the amino acid at position 252 is Phe, Ser, Thr, or Tyr;the amino acid at position 254 is Ser or Thr;the amino acid at position 255 is Arg, Gly, Ile, or Leu;the amino acid at position 256 is Ala, Arg, Asn, Asp, Gln, Glu, Pro, orThr;the amino acid at position 257 is Ala, Ile, Met, Asn, Ser, or Val;the amino acid at position 258 is Asp;the amino acid at position 260 is Ser;the amino acid at position 262 is Leu;the amino acid at position 270 is Lys;the amino acid at position 272 is Leu or Arg;the amino acid at position 279 is Ala, Asp, Gly, His, Met, Asn, Gln,Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 283 is Ala, Asp, Phe, Gly, His, Ile, Lys,Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;the amino acid at position 285 is Asn;the amino acid at position 286 is Phe;the amino acid at position 288 is Asn or Pro;the amino acid at position 293 is Val;the amino acid at position 307 is Ala, Glu, Gln, or Met;the amino acid at position 308 is Ile, Pro, or Thr;the amino acid at position 309 is Pro;the amino acid at position 311 is Ala, Glu, Ile, Lys, Leu, Met, Ser,Val, or Trp;the amino acid at position 312 is Ala, Asp, or Pro;the amino acid at position 314 is Ala or Leu;the amino acid at position 316 is Lys;the amino acid at position 317 is Pro;the amino acid at position 318 is Asn or Thr;the amino acid at position 332 is Phe, His, Lys, Leu, Met, Arg, Ser, orTrp;the amino acid at position 339 is Asn, Thr, or Trp;the amino acid at position 341 is Pro,the amino acid at position 343 is Glu, His, Lys, Gln, Arg, Thr, or Tyr;the amino acid at position 375 is Arg;the amino acid at position 376 is Gly, Ile, Met, Pro, Thr, or Val;the amino acid at position 377 is Lys;the amino acid at position 378 is Asp, Asn, or Val;the amino acid at position 380 is Ala, Asn, Ser, or Thr;the amino acid at position 382 is Phe, His, Ile, Lys, Leu, Met, Asn,Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 385 is Ala, Arg, Asp, Gly, His, Lys, Ser, orThr;the amino acid at position 386 is Arg, Asp, Ile, Lys, Met, Pro, Ser, orThr;the amino acid at position 387 is Ala, Arg, His, Pro, Ser, or Thr;the amino acid at position 389 is Asn, Pro, or Ser;the amino acid at position 423 is Asn;the amino acid at position 427 is Asn;the amino acid at position 428 is Leu, Met, Phe, Ser, or Thr;the amino acid at position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr;the amino acid at position 431 is His or Asn;the amino acid at position 433 is Arg, Gln, His, Ile, Lys, Pro, or Ser;the amino acid at position 434 is Ala, Gly, His, Phe, Ser, Trp, or Tyr;the amino acid at position 436 is Arg, Asn, His, Ile, Leu, Lys, Met, orThr;the amino acid at position 438 is Lys, Leu, Thr, or Trp;the amino acid at position 440 is Lys; andthe amino acid at position 442 is Lys, according to EU numbering.

Alterations that can enhance the binding to human FcRn under an acidicpH range condition as compared to human IgG include, for example, atleast one or more alterations selected from the following group:

an alteration comprising the amino acid at position 308 is Ile, theamino acid at position 309 is Pro, and/or the amino acid at position 311is Glu;an alteration comprising the amino acid at position 308 is Thr, theamino acid at position 309 is Pro, the amino acid at position 311 isLeu, the amino acid at position 312 is Ala, and/or the amino acid atposition 314 is Ala;an alteration comprising the amino acid at position 308 is Ile or Thr,the amino acid at position 309 is Pro, the amino acid at position 311 isGlu, Leu, or Ser, the amino acid at position 312 is Ala, and/or theamino acid at position 314 is Ala or Leu; andan alteration comprising the amino acid at position 308 is Thr, theamino acid at position 309 is Pro, the amino acid at position 311 isSer, the amino acid at position 312 is Asp, and/or the amino acid atposition 314 is Leu according to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example: an alteration comprising the amino acid atposition 251 is Leu, the amino acid at position 252 is Tyr, the aminoacid at position 254 is Ser or Thr, the amino acid at position 255 isArg, and/or the amino acid at position 256 is Glu according to EUnumbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 428 is Leu, Met,Phe, Ser, or Thr, the amino acid at position 433 is Arg, Gln, His, Ile,Lys, Pro, or Ser, the amino acid at position 434 is His, Phe, or Tyr,and/or the amino acid at position 436 is Arg, Asn, His, Lys, Met, orThr; andan alteration comprising the amino acid at position 428 is His or Met,and/or the amino acid at position 434 is His or Met according to EUnumbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 385 is Arg, theamino acid at position 386 is Thr, the amino acid at position 387 isArg, and/or the amino acid at position 389 is Pro; and

an alteration comprising the amino acid at position 385 is Asp, theamino acid at position 386 is Pro, and/or the amino acid at position 389is Ser according to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 250 is Gln or Glu;andan alteration comprising the amino acid at position 428 is Leu or Pheaccording to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 250 is Gln, and/orthe amino acid at position 428 is Leu or Phe; andan alteration comprising the amino acid at position 250 is Glu, and/orthe amino acid at position 428 is Leu or Phe according to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 307 is Gln and theamino acid at position 434 is Ala or Ser;an alteration comprising the amino acid at position 308 is Pro and theamino acid at position 434 is Ala;an alteration comprising the amino acid at position 252 is Tyr and theamino acid at position 434 is Ala;an alteration comprising the amino acid at position 378 is Val and theamino acid at position 434 is Ala;an alteration comprising the amino acid at position 428 is Leu and theamino acid at position 434 is Ala;an alteration comprising the amino acid at position 434 is Ala and theamino acid at position 436 is Ile;an alteration comprising the amino acid at position 308 is Pro and theamino acid at position 434 is Tyr; andan alteration comprising the amino acid at position 307 is Gln and theamino acid at position 436 is Ile according to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 307 is Gln, theamino acid at position 380 is Ala, and the amino acid at position 434 isSer;an alteration comprising the amino acid at position 307 is Gln, theamino acid at position 380 is Ala, and the amino acid at position 434 isAla;an alteration comprising the amino acid at position 252 is Tyr, theamino acid at position 308 is Pro, and the amino acid at position 434 isTyr; andan alteration comprising the amino acid at position 251 is Asp, theamino acid at position 307 is Gln, and the amino acid at position 434 isHis according to EU numbering.

In addition to the above, alterations that can enhance the binding tohuman FcRn under an acidic pH range condition as compared to human IgGinclude, for example, at least one or more alterations selected from thefollowing group:

an alteration comprising the amino acid at position 257 is Ile and theamino acid at position 311 is Ile;an alteration comprising the amino acid at position 257 is Ile and theamino acid at position 434 is His; andan alteration comprising the amino acid at position 376 is Val and theamino acid at position 434 is His according to EU numbering.

When an antigen-binding molecule of the present invention is producedusing the Fc region of human IgG, an antigen-binding molecule containingthe Fc region of IgG of the same subclass can be used as a control toassess the effect of the antigen-binding molecule of the presentinvention. Appropriate human IgG Fc regions that serve as a controlinclude the Fc regions of human IgG1 (SEQ ID NO: 49, which results fromadding A to the N terminus of RefSeq accession number AAC82527.1), humanIgG2 (SEQ ID NO: 50, which results from adding A to the N terminus ofRefSeq accession number AAB59393.1), human IgG3 (SEQ ID NO: 51, RefSeqaccession number CAA27268.1), and human IgG4 (SEQ ID NO: 52, whichresults from adding A to the N terminus of RefSeq accession numberAAB59394.1).

In mice, four types of Fcγ receptors have been identified: FcγRI (CD64),FcγRIIb (CD32), FcγRIII (CD16), and FcγRIV (CD16-2 or FcγRIII-2). As isthe case in humans, FcγRIIb is believed to be the sole inhibitory Fcγreceptor. FcγRIIb1 and FcγRIIb2 are reported to be splicing variants ofFcγRIIb. Both human and mouse FcγRIIb1 have a longer intracellulardomain than FcγRIIb2. FcγRIIb1 is confirmed to be expressed in B cells.FcγRIIb2 is confirmed to be expressed in macrophages, mast cells,dendritic cells, basophils, neutrophils, and eosinophils (J. Clin.Immunol. (2005) 25 (1), 1-18).

To date, functional deficiency or reduced expression of FcγRIIb has beenreported to have a correlation with the onset of autoimmune diseases inhumans. For example, it has been reported that in some SLE patients, thebinding of transcription activation factors is impaired due to theeffect of genetic polymorphism in the expression promoter region ofFcγRIIb, and the expression level of FcγRIIb is reduced (Hum. Genet.(2005) 117, 220-227; J. Immunol. (2004) 172, 7192-7199; J. Immunol.(2004) 172, 7186-7191). Furthermore, it has been reported that in someSLE patients, two types of genetic polymorphism of FcγRIIb are found inwhich the amino acid at position 233 is Ile or Thr. It has been reportedthat this site is located within the transmembrane domain of FcγRIIb,and in comparison to Ile, when the amino acid at position 233 is Thr, itbecomes difficult for FcγRIIb to be present on lipid rafts, resulting inimpairment of the signaling function of FcγRIIb (Nat. Med. (2005) 11,1056-1058; Hum. Mol. Genet., (2005) 14, 2881-2892). Regarding mice,knockout C57BL/6 mice with the FcγRIIb gene disrupted have been reportedto develop SLE-like symptoms such as autoantibody production andglomerulonephritis (Immunity 13 (2000) 277-285; J. Exp. Med. (2002) 195,1167-1174). In addition, it has been reported that the expression levelof FcγRIIb is reduced in mice that have been regarded as a spontaneousSLE onset model (Immunogenetics (2000) 51, 429-435; Int. Immunol. (1999)11, 1685-1691; Curr. Biol. (2000) 10, 227-230; J. Immunol. (2002) 169,4340-4346). These findings suggest that FcγRIIb regulates the humoralimmunity in mice as is the case in humans.

When an antibody that has an Fc region of the present inventioneliminates antigens via FcγRIIb, among the functions of FcγRIIb, theendocytotic function is thought to make the most important contribution.As described above, there are splicing variants of FcγRIIb: FcγRIIb1 andFcγRIIb2. It has been reported that the latter is primarily involved inthe endocytosis of the immune complex between antibody and antigen (J.Immunol. (1994), 152 574-585; Science (1992) 256, 1808-1812; Cell (1989)58, 317-327). To date, mouse FcγRIIb2 is reported to initiateendocytosis when incorporated into clathrin-coated pits (Cell (1989) 58,317-327). Meanwhile, it has been reported that a dileucine motif isrequired for the FcγRIIb2-mediated endocytosis, and the dileucine motifis conserved in both humans and mice (EMBO J. (1994) 13 (13),2963-2969). This finding also suggests that, like mouse, human FcγRIIb2has endocytotic ability.

On the other hand, unlike FcγRIIb2, FcγRIIb1 is reported not to induceendocytosis. In its intracellular domain, FcγRIIb1 has an insertionsequence which is not found in FcγRIIb2. The sequence is believed toinhibit the incorporation of FcγRIIb1 into clathrin-coated pits,resulting in inhibition of endocytosis (J. Cell. Biol. (1992) 116,875-888; J. Cell. Biol. (1989) 109, 3291-3302). As in mouse, humanFcγRIIb1 contains the insertion sequence, and thus due to a similarmechanism, there expects to be a difference in the endocytotic abilityof FcγRIIb1 and FcγRIIb2. Meanwhile, it has been reported that about 40%of the immune complexes on the cell surface are incorporated into cellsin 20 minutes both in humans and in mice (Mol. Immunol. (2011) 49,329-337; Science (1992) 256, 1808-1812). This finding suggests that inhumans, FcγRIIb2 internalizes immune complexes into cells at a ratesimilar to that in mice.

In the Fcγ receptor family, FcγRIIb alone has ITIM inside the cell inboth mice and humans, and the distribution of cells expressing FcγRIIbis identical. Thus, its function in the immunological regulation is alsoassumed to be the same. Furthermore, in light of the fact that immunecomplexes are taken up into cells at the same rate in humans and mice,the effect of FcγRIIb-mediated antigen elimination by antibody in humanis expected to be predictable by using mice. In fact, as shown in theExamples discussed below, as compared to when mIgG1 is administered,antigen clearance is increased when altered molecules (mF44 and mF46)with increased affinity for mouse FcγRIIb and FcγRIII relative to mIgG1are administered to normal mice.

Furthermore, as shown in the Examples described below, similarexperiments were carried out using Fc receptor γ chain-deficient mice.As for mice, it has been reported that FcγRs other than FcγRIIb areexpressed only in the co-presence of the γ chain. For this reason, Fcreceptor γ chain-deficient mice express FcγRIIb alone. The effect onantigen elimination produced when the binding activity to FcγRIIb isselectively increased can be studied by administering mF44 and mF46 toFc receptor γ chain-deficient mice. The results described in theExamples demonstrate that mF44 and mF46 administered to Fc receptor γchain-deficient mice increase antigen clearance as compared to whenmIgG1 is administered to the same mice. Furthermore, the resultsdescribed in the Examples demonstrate that even when administered to Fcreceptor γ chain-deficient mice, mF44 and mF46 eliminate antigens toalmost the same level as that when administered to normal mice.

Furthermore, as shown in the Examples described below, similarexperiments were carried out using FcγRIII-deficient mice. mIgG1, mF44,and mF46 bind to only FcγRIIb and FcγRIII among mouse FcγRs. For thisreason, the effect on antigen elimination produced when the bindingactivity to FcγRIIb is selectively increased can be studied byadministering these antibodies to FcγRIII-deficient mice. The resultsdescribed in the Examples demonstrate that when administered toFcγRIII-deficient mice, mF44 and mF46 increase antigen clearance ascompared to when mIgG1 is administered to the same mice. Furthermore,the results described in the Examples demonstrate that even whenadministered to FcγRIII-deficient mice, mF44 and mF46 eliminate antigensto almost the same level as that when administered to normal mice or Fcreceptor γ chain-deficient mice.

The results described above reveals that antigen elimination can beaccelerated by increasing the binding activity in an FcγRIIb-selectivemanner without increasing the binding activity to activating Fcγreceptors.

In addition to previous literature reports studied above, the results ofstudies using mice described above suggest that as is the case in mouse,FcγRIIb-mediated intake of immune complexes into cells occurs in humans,and as a result, antibodies that have an Fc region with increasedbinding activity to human FcγRIIb in a selective manner can accelerateantigen elimination. Furthermore, as discussed above, since the rate ofFcγRIIb-mediated intake of immune complexes into cells is assumed to becomparable between mouse and human, the antigen elimination-acceleratingeffect comparable to that of antibodies having an Fc region withincreased affinity for mouse FcγRIIb can be achieved with antibodiesthat have an Fc region with increased affinity for human FcγRIIb.

In general, the Kabat numbering system is used to describe residues inthe antibody variable regions (roughly, the residues at positions 1 to107 in the light chain, and the residues at positions 1 to 113 in theheavy chain) (for example, Kabat et al., Sequences of Proteins ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). “The EU numbering system”or “EU index” is generally used when referring to residues in the heavychain constant region of an antibody (for example, the EU index reportedin Kabat et al., supra). “The EU index of Kabat” refers to residuenumbering for human IgG1 EU antibody. In the present specification,unless otherwise specified, the residue numbers in antibody variableregions are described using the Kabat numbering system. In the presentspecification, unless otherwise specified, the residue numbers inantibody constant regions are described using the EU numbering system(see, for example, WO2006/073941).

Antigen-binding domains of the present invention have antigen-bindingactivity which is different between intracellular condition andextracellular condition. Intracellular condition and extracellularcondition refer to conditions that are different between in and outsideof the cell. Categories of conditions include, for example, ionconcentration, more specifically, hydrogen ion concentration (pH) andcalcium ion concentration. Intracellular condition preferably refers toan environment characteristic to the environment inside the endosome,while extracellular condition preferably refers to an environmentcharacteristic to the environment in plasma.

Antigen-binding domains with the property of having an antigen-bindingactivity that changes according to ion concentration can be obtained byscreening a large number of antigen-binding domains for domains havingsuch property. For example, when antigen-binding molecules of thepresent invention are antibodies, antibodies with the above-describedproperty can be obtained by producing a large number of antibodies whosesequences are different from one another by a hybridoma method or anantibody library method and measuring their antigen-binding activitiesat different ion concentrations. The B cell cloning method illustratedin Example 1 of the present specification is particularly suitable as amethod of screening for such antibodies. Furthermore, as describedbelow, at least one distinctive amino acid residue that can confer anantigen-binding domain with the property of having an antigen-bindingactivity that changes according to ion concentration is specified, toprepare as a library of a large number of antigen-binding domains thathave different sequences while sharing the distinctive amino acidresidues as a common structure. Such a library can be screened toefficiently isolate antigen-binding domains that have the propertydescribed above.

In an embodiment of the present invention, the condition of ionconcentrations refers to the condition of hydrogen ion concentrations orpH condition. In the present invention, the concentration of proton,i.e., the nucleus of hydrogen atom, is treated as synonymous withhydrogen ion concentration index (pH). When the activity of hydrogen ionin an aqueous solution is represented as aH+, pH is defined as −log10aH+. When the ionic strength of the aqueous solution is low (forexample, lower than 10⁻³), aH+ is nearly equal to the hydrogen ionstrength. For example, the ionic product of water at 25° C. and 1atmosphere is Kw=aH+aOH=10⁻¹⁴, and therefore in pure water,aH+=aOH=10⁻⁷. In this case, pH=7 is neutral; an aqueous solution whosepH is lower than 7 is acidic or whose pH is greater than 7 is alkaline.

In the present invention, when the pH condition is used as the ionconcentration condition, it is desirable that the antigen-bindingactivity under an acidic pH range (i.e., a high hydrogen ionconcentration or low pH) condition is lower than that under a neutral pHrange (i.e., a low hydrogen ion concentration or high pH) condition.

Intracellular pH is acidic as compared to extracellular pH. Conversely,extracellular pH is neutral as compared to intracellular pH. The presentinvention provides antigen-binding molecules in which the extracellularcondition is a neutral pH range condition and the intracellularcondition is an acidic pH range condition. In the present invention, theacidic pH range is preferably pH 4.0 to pH 6.5, more preferably pH 5.0to pH 6.5, still more preferably any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably pH 5.8 to pH6.0 which is close to the pH in the early endosome in vivo. Meanwhile,in the present invention, the neutral pH range is preferably pH 6.7 topH 10.0, more preferably pH 7.0 to pH 9.0, still more preferably any ofpH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, andparticularly preferably pH 7.4 which is close to the pH in plasma (inblood).

When the level of antigen-binding activity is compared between theacidic pH range condition and the neutral pH range condition, it ispreferable that the binding of antigen-binding domains of the presentinvention is stronger under a neutral pH range condition than under anacidic pH range condition. When the level of binding activity isexpressed with the dissociation constant (KD), the value of KD (acidicpH)/KD (neutral pH) is preferably 2 or more, more preferably 10 or more,and still more preferably 40 or more. The upper limit of the value of KD(acidic pH)/KD (neutral pH) is not particularly limited, and may be anyvalue such as 100, 400, 1000, or 10000, as long as it can be producedwith the techniques of skilled artisans. It is possible to use thedissociation rate constant (kd) instead of KD. When it is difficult tocalculate the KD value, the activity may be assessed based on the levelof binding response in Biacore when analytes are passed at the sameconcentration. When antigens are passed over a chip immobilized withantigen-binding molecules of the present invention, the binding responseunder an acidic pH range condition is preferably ½ or less of thebinding response under a neutral pH range condition, more preferably ⅓or less, still more preferably ⅕ or less, and particularly preferably1/10 or less.

It is known that in general the in vivo extracellular pH (for example,in plasma) is neutral while the intracellular pH (for example, in theendosome) is acidic. When the property of having a lower antigen-bindingactivity under an intracellular pH condition than under an extracellularpH condition is conferred to the antigen-binding domain ofantigen-binding molecules of the present invention, antigens that havebound to the antigen-binding molecule of the present invention outsideof the cell dissociate from the antigen-binding molecule of the presentinvention inside the cell, thereby enhancing antigen incorporation intothe cell from the outside of the cell. Such antigen-binding molecules,when administered to the living body, can reduce antigen concentrationin plasma and thereby reduce the physiological activity of antigens invivo. Thus, antigen-binding molecules of the present invention areuseful.

Methods for conferring to antigen-binding domains of the presentinvention the property of binding more weakly to antigens under anacidic pH range condition than binding under a neutral pH rangecondition are not particularly limited, and the property may beconferred by any methods. Specifically, the methods are described inWO2009/125825, and include a method of substituting at least one aminoacid residue with histidine and/or inserting at least one histidine inan antigen-binding domain. In a preferred embodiment, theantigen-binding molecules of the present invention contain asubstitution of at least one amino acid residue by histidine and/or aninsertion of at least one histidine in their antigen-binding domain. Itis already known that the pH-dependent antigen-binding activity can beconferred to antibodies by substituting their amino acid residues withhistidine (Ito W et al., FEBS Lett. (1992) 309, 85-88). The site ofhistidine substitution and/or insertion is not particularly limited, andamino acid residues at any positions may be substituted with histidine,and histidine may be inserted at any sites as long as theantigen-binding activity under an acidic pH range condition is reducedto be lower than that under a neutral pH range condition. Histidinesubstitution and/or insertion sites preferably include, for example, thesite to which an antigen directly binds and portions that contribute tothe preservation of the conformation of the site. For example, when theantigen-binding domain is an antibody variable region, the sites includeCDRs and regions that contribute to the preservation of theirconformation. Specifically, the sites include H27, H31, H32, H33, H35,H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102 in theheavy chain; and L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94 inthe light chain. Among these sites, H32, H61, L53, L90, and L94 areexpected to be high in universality (the positions of amino acidresidues are indicated according to Kabat numbering (Kabat E A et al.,1991. Sequences of Proteins of Immunological Interest. NIH)). Preferredcombinations of substituting multiple sites with histidine include, forexample, the combination of H27, H31, and H35; the combination of H27,H31, H32, H35, H58, H62, and H102; the combination of L32 and L53; andthe combination of L28, L32, and L53. Furthermore, preferredcombinations of substitution sites in the heavy chain and light chaininclude, for example, the combination of H27, H31, L32, and L53.

Histidine is substituted and/or inserted at random into theantigen-binding domains of antigen-binding molecules of the presentinvention by a method such as histidine scanning, where histidine isused instead of alanine in an alanine scanning method known to thoseskilled in the art. It is possible to select from the resultingmolecules, antigen-binding domains that bind more weakly to antigensunder an acidic pH range condition than under a neutral pH rangecondition.

The number of histidines to be substituted and/or inserted can beappropriately determined by those skilled in the art. Histidine may besubstituted or inserted at only one site. Alternatively, histidine maybe substituted or inserted at two or more sites. Alternatively,histidine substitution and histidine insertion may be combined at two ormore sites.

It is preferable that the antigen-binding domains have comparableantigen-binding activities before and after histidine substitutionand/or insertion. Herein, comparable activity means 10% or more,preferably 30% or more, more preferably 50% or more, still morepreferably 80% or more, and particularly preferably 90% or more of theoriginal activity.

When antigen-binding molecules provided by the present invention containthe antibody constant region, methods for conferring to theantigen-binding provided by the present invention the property ofbinding more weakly to the antigen under an acidic pH range conditionthan under a neutral pH range condition include methods for altering theantibody constant region. Methods for altering the antibody constantregion include, for example, methods in which the constant region iscompared among isotypes (IgG1, IgG2, IgG3, and IgG4) to select anisotype with reduced antigen-binding activity under an acidic pH rangecondition (dissociation rate is accelerated under an acidic pH rangecondition). Such methods also include methods that reduce the antigenbinding activity under an acidic pH range condition (dissociation rateis accelerated under an acidic pH range condition) by introducing aminoacid alteration into the amino acid sequence of an isotype (IgG1, IgG2,IgG3, or IgG4). The sequence of the hinge domain of an antibody constantregion varies considerably among isotypes (IgG1, IgG2, IgG3, and IgG4).The difference in the amino acid sequence of the hinge domain has greateffects on the antigen-binding activity. Thus, the antigen-bindingactivity under an acidic pH range condition can be reduced by selectingan isotype appropriate for the type of binding antigen. Meanwhile, whenan amino acid alteration is introduced into the amino acid sequence ofan isotype, the site of introducing an amino acid alteration ispreferably within the hinge domain.

A large number of antigen-binding domains is prepared as a library, inwhich the antigen-binding domains have different sequences while sharingas a common structure the above-described amino acid residues thatchange the antigen-binding activity according to the hydrogen ionconcentration. The library can be screened to efficiently obtainantigen-binding domains with binding activity to a desired antigen, andin which their antigen-binding activity changes according to thehydrogen ion concentration.

For example, a light chain variable region with a framework sequencecontaining at least one amino acid residue that changes theantigen-binding activity according to the hydrogen ion concentration canbe combined with a heavy chain variable region containing a randomsequence, to construct a library that contains multiple antigen-bindingdomains that have different sequences while sharing as a commonstructure amino acid residues that change the antigen-binding activityaccording to the hydrogen ion concentration. In a preferred embodimentwhere such amino acid residues are introduced into the light chainaltered region, the amino acid residues may be contained in the CDR1 ofthe light chain altered region, more preferably at positions 24, 27, 28,31, 32, and/or 34 according to Kabat numbering in the CDR1 of the lightchain variable region. In another preferred embodiment, the amino acidresidues may be contained in the CDR2 of the light chain variableregion, more preferably at positions 50, 51, 52, 53, 54, 55, and/or 56according to Kabat numbering in the CDR2 of the light chain variableregion. In still another preferred embodiment, the amino acid residuesmay be contained in the CDR3 of the light chain variable region, morepreferably at positions 89, 90, 91, 92, 93, 94, and/or 95A according toKabat numbering in the CDR3 of the light chain variable region. Theamino acid residues may be contained alone, or two or more may becontained in combination as long as they change the antigen-bindingactivity according to the hydrogen ion concentration.

When the light chain variable region containing at least one amino acidresidue that changes the antigen-binding activity according to thehydrogen ion concentration is combined with the heavy chain variableregion containing a random sequence to produce an antigen-bindingdomain, it is possible to design it in such a way that its light chainvariable region further contains flexible residues. Such flexibleresidues are not particularly limited in number and position, as long asthe antigen-binding activity of the antigen-binding domain of thepresent invention is changed according to the hydrogen ionconcentration. Specifically, the light chain CDR sequences and/or FRsequences may contain one or more flexible residues. Without beingparticularly limited thereto, flexible residues that are introduced intothe sequence of the light chain variable region include, for example,amino acid residues shown in Tables 7 and 8. Meanwhile, without beingparticularly limited thereto, the sequence of the light chain variableregion, other than flexible residues and amino acid residues that changethe antigen-binding activity according to the hydrogen ionconcentration, preferably includes germ-line sequences such as Vk1 (SEQID NO: 58), Vk2 (SEQ ID NO: 59), Vk3 (SEQ ID NO: 60), and Vk4 (SEQ IDNO: 61).

TABLE 7 POSITION AMINO ACID CDR1 28 S: 100% 29 I: 100% 30 N: 25% S: 25%R: 25% H: 25% 31 S: 100% 32 H: 100% 33 L: 100% 34 A: 50% N: 50% CDR2 50H: 100% OR A: 25% D: 25% G: 25% K: 25% 51 A: 100% A: 100% 52 S: 100% S:100% 53 K: 33.3% N: 33.3% S: 33.3% H: 100% 54 L: 100% L: 100% 55 Q: 100%Q: 100% 56 S: 100% S: 100% CDR3 90 Q: 100% OR Q: 100% 91 H: 100% S:33.3% R: 33.3% Y: 33.3% 92 G: 25% N: 25% S: 25% Y: 25% H: 100% 93 H:33.3% N: 33.3% S: 33.3% H: 33.3% N: 33.3% S: 33.3% 94 S: 50% Y: 50% S:50% Y: 50% 95 P: 100% P: 100% 96 L: 50% Y: 50% L: 50% Y: 50% (“POSITION”indicates Kabat numbering)

TABLE 8 CDR POSITION AMINO ACID CDR1 28 S: 100% 29 I: 100% 30 H: 30% N:10% S: 50% R: 10% 31 N: 35% S: 65% 32 H: 40% N: 20% Y: 40% 33 L: 100% 34A: 70% N: 30% CDR2 50 A: 25% D: 15% G: 25% H: 30% K: 5% 51 A: 100% 52 S:100% 53 H: 30% K: 10% N: 15% S: 45% 54 L: 100% 55 Q: 100% 56 S: 100%CDR3 90 Q: 100% 91 H: 30% S: 15% R: 10% Y: 45% 92 G: 20% H: 30% N: 20%S: 15% Y: 15% 93 H: 30% N: 25% S: 45% 94 S: 50% Y: 50% 95 P: 100% 96 L:50% Y: 50% (“POSITION” indicates Kabat numbering)

Any amino acid residues may be suitably used as an amino acid residuethat changes the antigen-binding activity according to the hydrogen ionconcentration. Specifically, such amino acid residues include those witha side-chain pKa of 4.0 to 8.0. Such electron-donating amino acidspreferably include, for example, native amino acids such as histidineand glutamic acid, and unnatural amino acids such as histidine analogs(US20090035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768).The amino acid residues particularly preferably include, for example,those with a side-chain pKa of 6.0 to 7.0. Such electron-donating aminoacids preferably include, for example, histidine.

Furthermore, in one embodiment of the present invention, the ionconcentration refers to a metal ion concentration. “Metal ions” refer toions of group I elements except hydrogen such as alkaline metals andcopper group elements, group II elements such as alkaline earth metalsand zinc group elements, group III elements except boron, group IVelements except carbon and silicon, group VIII elements such as irongroup and platinum group elements, elements belonging to subgroup A ofgroups V, VI, and VII, and metal elements such as antimony, bismuth, andpolonium. Metal atoms have the property of releasing valence electronsto become cations. This is referred to as ionization tendency. Metalswith strong ionization tendency are deemed to be chemically active.

In the present invention, preferred metal ions include, for example,calcium ion. Calcium ion is involved in modulation of many biologicalphenomena, including contraction of muscles such as skeletal, smooth,and cardiac muscles; activation of movement, phagocytosis, and the likeof leukocytes; activation of shape change, secretion, and the like ofplatelets; activation of lymphocytes; activation of mast cells includingsecretion of histamine; cell responses mediated by catecholamine areceptor or acetylcholine receptor; exocytosis; release of transmittersubstances from neuron terminals; and axoplasmic flow in neurons. Knownintracellular calcium ion receptors include troponin C, calmodulin,parvalbumin, and myosin light chain, which have several calciumion-binding sites and are believed to be derived from a common origin interms of molecular evolution. There are also many known calcium-bindingmotifs. Such well-known motifs include, for example, cadherin domains,EF-hand of calmodulin, C2 domain of Protein kinase C, Gla domain ofblood coagulation protein Factor IX, C-type lectins ofacyaroglycoprotein receptor and mannose-binding receptor, A domains ofLDL receptors, annexin, thrombospondin type 3 domain, and EGF-likedomains.

In the present invention, when the metal ion is calcium ion, it isdesirable that the antigen-binding activity is lower under a low calciumion concentration condition than under a high calcium ion concentrationcondition.

Meanwhile, the intracellular calcium ion concentration is lower than theextracellular calcium ion concentration. Conversely, the extracellularcalcium ion concentration is higher than the intracellular calcium ionconcentration. In the present invention, the low calcium ionconcentration is preferably 0.1 μM to 30 μM, more preferably 0.5 μM to10 μM, and particularly preferably 1 μM to 5 μM which is close to thecalcium ion concentration in the early endosome in vivo. Meanwhile, inthe present invention, the high calcium ion concentration is preferably100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularlypreferably 0.5 mM to 2.5 mM which is close to the calcium ionconcentration in plasma (in blood). In the present invention, it ispreferable that the low calcium ion concentration is the calcium ionconcentration in endosomes, and the high calcium ion concentration isthe calcium ion concentration in plasma.

When the level of antigen-binding activity is compared between low andhigh calcium ion concentrations, it is preferable that the binding ofantigen-binding domains of the present invention is stronger at a highcalcium ion concentration than at a low calcium ion concentration. Inother words, it is preferable that the antigen-binding activity ofantigen-binding domains of the present invention is lower at a lowcalcium ion concentration than at a high calcium ion concentration.

When the level of binding activity is expressed with the dissociationconstant (KD), the value of KD (low calcium ion concentration)/KD (highcalcium ion concentration) is greater than 1, preferably 2 or more,still more preferably 10 or more, and yet more preferably 40 or more.The upper limit of the value of KD (low calcium ion concentration)/KD(high calcium ion concentration) is not particularly limited, and may beany value such as 100, 400, 1000, or 10000, as long as it can beproduced with the techniques of skilled artisans. It is possible to usethe dissociation rate constant (kd) instead of KD. When it is difficultto calculate the KD value, the activity may be assessed based on thelevel of binding response in Biacore when analytes are passed at thesame concentration. When antigens are passed over a chip immobilizedwith antigen-binding molecules of the present invention, the bindingresponse at a low calcium concentration is preferably ½ or less of thebinding response at a high calcium concentration, more preferably ⅓ orless, still more preferably ⅕ or less, and particularly preferably 1/10or less.

It is known that in general the in vivo extracellular calcium ionconcentration (for example, in plasma) is high, and the intracellularcalcium ion concentration (for example, in the endosome) is low. Thus,in the present invention, it is preferable that the extracellularcondition is a high calcium ion concentration, and the intracellularcondition is a low calcium ion concentration.

When the property that the antigen-binding activity is lower under anintracellular calcium ion concentration condition than under anextracellular calcium ion concentration condition is conferred to theantigen-binding domain of antigen-binding molecules of the presentinvention, antigens that have bound to antigen-binding molecule of thepresent invention outside of the cell dissociate from theantigen-binding molecule of the present invention inside the cell,thereby enhancing antigen incorporation into the cell from the outsideof the cell. Such antigen-binding molecules, when administered to theliving body, can reduce antigen concentration in plasma and reduce thephysiological activity of antigens in vivo. Thus, antigen-bindingmolecules of the present invention are useful.

Methods of screening for antigen-binding domains or antigen-bindingmolecules having a lower antigen-binding activity under a low calciumion concentration condition than under a high calcium ion concentrationcondition include, for example, the method described in WO2012/073992(for example, paragraphs 0200-0213).

Methods for conferring antigen-binding domains of the present inventionwith the property of binding more weakly to antigens under a low calciumion concentration condition than under a high calcium ion concentrationcondition are not particularly limited, and may be carried out by anymethods. Specifically, the methods are described in Japanese PatentApplication No. 2011-218006 and include, for example, methods forsubstituting at least one amino acid residue in an antigen-bindingdomain with an amino acid residue having metal chelating activity,and/or inserting into an antigen-binding domain at least one amino acidresidue having metal chelating activity. Antigen-binding molecules ofthe present invention in which at least one amino acid residue of theantigen-binding domain has been substituted with an amino acid residuehaving metal chelating activity and/or at least one amino acid residuehaving metal chelating activity has been inserted into theantigen-binding domain are a preferred embodiment of antigen-bindingmolecules of the present invention. Amino acid residues having metalchelating activity preferably include, for example, serine, threonine,asparagine, glutamine, aspartic acid, and glutamic acid.

Furthermore, amino acid residues that change the antigen-bindingactivity of antigen-binding domains according to the calcium ionconcentration preferably include, for example, amino acid residues thatform a calcium-binding motif. Calcium-binding motifs are well known tothose skilled in the art, and have been described in detail (forexample, Springer et al., (Cell (2000) 102, 275-277); Kawasaki andKretsinger (Protein Prof. (1995) 2, 305-490); Moncrief et al., (J. Mol.Evol. (1990) 30, 522-562); Chauvaux et al., (Biochem. J. (1990) 265,261-265); Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (NewBiol. (1990) 2, 410-419); Schaefer et al., (Genomics (1995) 25, 638 to643); Economou et al., (EMBO J. (1990) 9, 349-354); Wurzburg et al.,(Structure. (2006) 14, 6, 1049-1058)). EF hand in troponin C,calmodulin, parvalbumin, and myosin light chain; C2 domain in proteinkinase C; Gla domain in blood coagulation protein factor IX; C-typelectin of acyaroglycoprotein receptor and mannose-binding receptor,ASGPR, CD23, and DC-SIGN; A domain in LDL receptor; annexin domain;cadherin domain; thrombospondin type 3 domain; and EGF-like domain arepreferably used as calcium-binding motifs. In addition to the above, thecalcium-binding motif in the antigen-binding domain of SEQ ID NO: 57 ispreferably used.

Antigen-binding domains of the present invention can contain amino acidresidues that change the antigen-binding activity according to thecalcium ion concentration, such as the above-described amino acidresidues with metal chelating activity and amino acid residues that forma calcium-binding motif. The location of such amino acid residues in theantigen-binding domain is not particularly limited, and they may belocated at any position as long as the antigen-binding activity changesaccording to the calcium ion concentration. Meanwhile, such amino acidresidues may be contained alone or in combination of two or more, aslong as the antigen-binding activity changes according to the calciumion concentration. The amino acid residues preferably include, forexample, serine, threonine, asparagine, glutamine, aspartic acid, andglutamic acid. When an antigen-binding domain is an antibody variableregion, the amino acid residues may be contained in the heavy chainvariable region and/or the light chain variable region. In a preferredembodiment, the amino acid residues may be contained in the CDR3 of theheavy chain variable region, more preferably at positions 95, 96, 100a,and/or 101 according to Kabat numbering in the CDR3 of the heavy chainvariable region.

In another preferred embodiment, the amino acid residues may becontained in the CDR1 of the light chain variable region, morepreferably at positions 30, 31, and/or 32 according to Kabat numberingin the CDR1 of the light chain variable region. In still anotherpreferred embodiment, the amino acid residues may be contained in theCDR2 of the light chain variable region, more preferably at position 50according to Kabat numbering in the CDR2 of the light chain variableregion. In yet another preferred embodiment, the amino acid residues maybe contained in the CDR3 of the light chain variable region, morepreferably at position 92 according to Kabat numbering in the CDR3 ofthe light chain variable region.

Furthermore, it is possible to combine the above-described embodiments.For example, the amino acid residues may be contained in two or threeCDRs selected from the CDR1, CDR2, and CDR3 of the light chain variableregion, more preferably at any one or more of positions 30, 31, 32, 50,and/or 92 according to Kabat numbering in the light chain variableregion.

A large number of antigen-binding domains that have different sequenceswhile sharing as a common structure the above-described amino acidresidues that change the antigen-biding activity according to thecalcium ion concentration are prepared as a library. The library can bescreened to efficiently obtain antigen-binding domains with bindingactivity to a desired antigen, in which their antigen-binding activitychanges according to the calcium ion concentration.

When the antigen-binding domain is an antibody variable region, in aparticularly preferred embodiment, it is desirable that the frameworksequences of the light chain and/or heavy chain variable region have ahuman germ-line framework sequence. Thus, in an embodiment of thepresent invention, when the framework sequence is completely a humansequence, the antigen-binding domain of the present invention, whenadministered to humans (for example, to treat diseases), is expected toinduce little or no immunogenic response. In this context, herein,“having a germ-line sequence” means that a portion of the frameworksequence of the present invention is identical to a portion of a humangerm-line framework sequence. For example, even when an antigen-bindingdomain of the present invention has a sequence resulting from combiningmultiple different human germ-line framework sequences, it is anantigen-binding domain of the present invention “having a germ-linesequence”.

Without being bound by a particular theory, one reason why the use of agerm-line sequence is expected to result in the elimination of adverseimmune responses in most individuals is believed to be as follows. As aresult of the process of affinity maturation during normal immuneresponses, somatic cell mutations occur frequently in the variableregion of immunoglobulins. Such mutations also affect residues in theframework region while they primarily occur around CDRs of whichsequences are hypervariable. There is no such framework mutation in thegerm line sequences, and thus they can be immunogenic in patients. Onthe other hand, the normal human population is exposed to most frameworksequences expressed from the germ line genes. As a result ofimmunotolerance, the germ line sequences are expected to be only weaklyimmunogenic or nonimmunogenic in patients. To maximize the possibilityof immunotolerance, genes encoding the variable region can be selectedfrom a group of functional germ-line genes (which are expressednormally).

The framework preferably includes, for example, currently knownframework region sequences that are completely of the human type. Thesequences of such framework regions are shown, for example, on thewebsites of V-Base (http://vbase.mrc-cpe.cam.ac.uk/). Those frameworkregion sequences can be appropriately used as a germ line sequencecontained in an antigen-binding domain of the present invention. Thegerm line sequences may be categorized according to their similarity(Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798); Williams andWinter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al. (Nat.Genetics (1994) 7, 162-168)). Appropriate germ line sequences can beselected from VK, which is grouped into seven subgroups; Vλ, which isgrouped into ten subgroups; and VH, which is grouped into sevensubgroups.

Fully human VH sequences preferably include, but are not limited to, forexample, VH sequences of:

subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45,VH1-46, VH1-58, and VH1-69);subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70);subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20,VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49,VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74);subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, andVH4-61);subgroup VH5 (VH5-51);subgroup VH6 (VH6-1); andsubgroup VH7 (VH7-4 and VH7-81).These are also described in known documents (Matsuda et al. (J. Exp.Med. (1998) 188, 1973-1975)) and such, and thus persons skilled in theart can appropriately design antigen-binding domains of the presentinvention based on the information of these sequences. It is alsopreferable to use other fully human framework regions or frameworksub-regions.

Fully human VK sequences preferably include, but are not limited to, forexample:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23,L24, O2, O4, O8, O12, O14, and O18, grouped into subgroup Vk1;A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped intosubgroup Vk2;A11, A27, L2, L6, L10, L16, L20, and L25, grouped into subgroup Vk3;B3, grouped into subgroup Vk4;B2 (herein also referred to as Vk5-2), grouped into subgroup Vk5; andA10, A14, and A26, grouped into subgroup VK6(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable andZachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022);Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human VL sequences preferably include, but are not limited to, forexample:

V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18,V1-19, V1-20, and V1-22, grouped into subgroup VL1;V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,grouped into subgroup VL1;V3-2, V3-3, and V3-4, grouped into subgroup VL3;V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into subgroup VL4; andV5-1, V5-2, V5-4, and V5-6, grouped into subgroup VL5 (Kawasaki et al.(Genome Res. (1997) 7, 250-261)).

Normally, these framework sequences are different from one another atone or more amino acid residues. These framework sequences can be usedin combination with at least one amino acid residue that alters theantigen-binding activity depending on ion concentrations describedabove. Other examples of frameworks include, but are not limited to, forexample, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (for example, Kabatet al. (1991) supra; Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

For example, a light chain variable region that contains in itsframework sequence at least one amino acid residue that changes theantigen-binding activity according to the calcium ion concentration canbe combined with a heavy chain variable region containing a randomsequence to construct a library containing a number of antigen-bindingdomains that have different sequences while sharing as a commonstructure the amino acid residues that change the antigen-bindingactivity according to the calcium ion concentration. Without beingparticularly limited thereto, a preferred example includes a library ofantigen-binding domains resulting from combining the heavy chainvariable region containing a random sequence with the light chainvariable region that belongs to the Vk5-2 family such as SEQ ID NO: 57(Vk5-2). Preferred examples also include a library of antigen-bindingdomains resulting from combining the heavy chain variable regioncontaining a random sequence with the sequence of a light chain variableregion in which at least one amino acid residue that changes theantigen-binding activity according to the calcium ion concentration hasbeen substituted for specific amino acid residues in a germ-linesequence such as SEQ ID NO: 58 (Vk1), SEQ ID NO: 59 (Vk2), SEQ ID NO: 60(Vk3), and SEQ ID NO: 61 (Vk4).

Furthermore, it is possible to design in such a way that flexibleresidues are contained in the light chain variable region of whichframework sequences contain at least one amino acid residue that changesthe antigen-binding activity according to the calcium ion concentration.Such flexible residues are not particularly limited in number andposition as long as the antigen-binding activity of antigen-bindingdomains of the present invention changes according to the ionconcentration. Specifically, the CDR and/or FR sequences of the heavychain variable region and/or the light chain variable region may containone or more flexible residues. Without being particularly limitedthereto, flexible residues that are introduced into the light chainvariable region of SEQ ID NO: 57 (Vk5-2) include, for example, aminoacid residues shown in Tables 9 and 10.

TABLE 9 Kabat CDR NUMBERING 70% AMINO ACID OF THE TOTAL CDR1 28 S: 100%29 I: 100% 30 E: 72% N: 14% S: 14% 31 D: 100% 32 D: 100% 33 L: 100% 34A: 70% N: 30% CDR2 50 E: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25% S:45% T: 25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25% S:15% R: 15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S: 50%R: 10% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%

TABLE 10 Kabat CDR NUMBERING 30% AMINO ACID OF THE TOTAL CDR1 28 S: 100%29 I: 100% 30 E: 83% S: 17% 31 D: 100% 32 D: 100% 33 L: 100% 34 A: 70%N: 30% CDR2 50 H: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25% S: 45% T:25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25% S: 15% R:15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S: 50% R: 10%94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%

Herein, flexible residue refers to an amino acid residue that is presentat a position where the type of amino acid varies greatly in the lightchain variable regions and heavy chain variable regions when comparingthe amino acid sequences of known and/or native antibodies orantigen-binding domains. The positions that vary greatly are generallypresent in the CDR regions. For example, the data provided as Kabat,Sequences of Proteins of Immunological Interest (National Institute ofHealth Bethesda Md.) (1987 and 1991) is useful to determine thepositions that vary greatly in known and/or native antibodies.Furthermore, various databases on the Internet(http://vbase.mrc-cpe.cam.ac.uk/,http://www.bioinf.org.uk/abs/index.html) provide the collected sequencesof many human light chains and heavy chains. The sequence information isuseful for determining the positions that vary greatly in the presentinvention. In the present invention, when at a certain amino acidposition, the number of possible variations of amino acids is preferablyabout 2 to 20, preferably about 3 to 19, preferably about 4 to 18,preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8to 14, preferably 9 to 13, and preferably 10 to 12, such a position isdefined as varying greatly. Meanwhile, at a certain position, the numberof possible variations of amino acids can be preferably at least about2, preferably at least about 4, preferably at least about 6, preferablyat least about 8, preferably at least about 10, and preferably at leastabout 12.

In order to produce an antigen-binding domain, when a light chainvariable region containing at least one amino acid residue that changesthe antigen-binding activity according to the calcium ion concentrationis combined with a heavy chain variable region containing a randomsequence, it is possible to design it in such a way that its light chainvariable region further contains flexible residues. The flexibleresidues are not particularly limited in number and position as long asthe antigen-binding activity changes according to the calcium ionconcentration. Specifically, the light chain CDR sequences and/or FRsequences may contain one or more flexible residues. Without beingparticularly limited, flexible residues that are introduced into thelight chain variable region include, for example, amino acid residuesshown in Tables 9 and 10.

In the present invention, known methods can be appropriately combined toprepare as a randomized variable region library the heavy chain and/orthe light chain variable region that have a random sequence. In anembodiment, immune libraries constructed based on antibody genes derivedfrom the lymphocytes of animals immunized with a specific antigen,humans whose antibody titer in blood has been increased due tovaccination, patients with infection, cancer patients, autoimmunedisease patients, and such may be suitably used as a randomized variableregion library.

In another embodiment, a synthetic library in which arbitrary CDRsequences of V genes from genomic DNA or functional reshaped V genes arereplaced with a set of synthetic oligonucleotides encoding codon sets ofan appropriate length can also be preferably used as a randomizedvariable region library. In this case, it is possible to replace CDR3sequences alone, since sequence polymorphism is observed in the CDR3 ofthe heavy chain variable region. When diversifying the amino acidsequence of an antigen-binding molecule, it is preferable to generatevariations in the amino acid residues at surface-exposed positions inthe antigen-binding molecule. Surface-exposed position refers to aposition where surface exposure and/or contact with an antigen isdetermined to be possible, based on the conformation, structuralensemble, and/or modeled structure of an antigen-binding molecule. Inthe variable region, such positions are generally the CDRs.Surface-exposed positions can be determined from the coordinates of athree dimensional model of the antigen-binding molecule using computerprograms such as the InsightII program (Accelrys). Surface-exposedpositions can also be determined using algorithms known in the art (forexample, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly(J. Appl. Cryst. (1983) 16, 548-558)). Alternatively, thesurface-exposed positions can be determined based on the information onthe three dimensional structure obtained from antibodies and softwaresuitable for protein modeling. Software that can be used for thispurpose preferably includes the SYBYL Biopolymer Module software (TriposAssociates). When the algorithm requires the input size parameter fromthe user, the “size” of probe for use in computation is generally set tobe about 1.4 Å or less in radius. Furthermore, a method for determiningthe surface-exposed region or area using PC software is described byPacios (Comput. Chem. (1994) 18 (4), 377-386; and J. Mol. Model. (1995)1, 46-53).

In still another embodiment, a naive library constructed from antibodygenes derived from lymphocytes of healthy persons can also beparticularly preferably used as a randomized variable region library(Gejima et al., (Human Antibodies (2002) 11, 121-129); and Cardoso etal., (Scand. J. Immunol. (2000) 51, 337-344)). Much variation isexpected in the repertoire of antibody sequences derived fromlymphocytes of healthy persons, because it is unbiased. In the presentinvention, an amino acid sequence containing a naive sequence refers toan amino acid sequence obtained from such a naive library.

In an embodiment of the present invention, antigen-binding domains ofthe present invention can be obtained from a library that contains anumber of antigen-binding domains with sequences different from oneanother, which is constructed by combining a light chain variable regionhaving a random sequence with a heavy chain variable region containingat least one amino acid residue that changes the antigen-bindingactivity according to the calcium ion concentration. Without beingparticularly limited thereto, the libraries preferably include, forexample, libraries of antigen-binding domains in which the heavy chainvariable region of SEQ ID NO: 117 (6RL#9-IgG1) or SEQ ID NO: 119(6KC4-1#85-IgG1) is combined with a light chain variable region having arandom sequence. Alternatively, a light chain variable region having agerm-line sequence may be suitably selected and used instead of thelight chain variable region having a random sequence. Without beingparticularly limited thereto, the libraries preferably include, forexample, libraries of antigen-binding domains in which the heavy chainvariable region of SEQ ID NO: 117 (6RL#9-IgG1) or SEQ ID NO: 119(6KC4-1#85-IgG1) is combined with the light chain variable region havinga germ-line sequence.

Furthermore, the above-described heavy chain variable region containingat least one amino acid residue that changes the antigen-bindingactivity according to the calcium ion concentration can be designed insuch a way that it additionally contains flexible residues. The flexibleresidues are not particularly limited in number and position, as long asthe antigen-binding activity of antigen-binding domains of the presentinvention changes according to the calcium ion concentration.Specifically, the CDR sequences and/or FR sequences of the heavy chainand/or the light chain may contain one or more flexible residues.Without being particularly limited thereto, flexible residues that areintroduced into the heavy chain variable region of SEQ ID NO: 117(6RL#9-IgG1) include, for example, the entire amino acid residues of theheavy chain CDR1 and the heavy chain CDR2, and amino acid residues ofthe heavy chain CDR3 except for at positions 95, 96, and/or 100a.Meanwhile, flexible residues that are introduced into the heavy chainvariable region of SEQ ID NO: 119 (6KC4-1#85-IgG1) include, for example,the entire amino acid residues of the heavy chain CDR1 and the heavychain CDR2, and amino acid residues of the heavy chain CDR3 except forat positions 95 and/or 101.

Alternatively, a library that contains multiple antigen-binding domainswith sequences different from one another can be constructed bycombining a light chain variable region having a random sequence or alight chain variable region having a germ-line sequence with theabove-described heavy chain variable region introduced with at least oneamino acid residue that changes the antigen-binding activity accordingto the calcium ion concentration. The libraries preferably include, forexample, libraries of antigen-binding domains in which a light chainvariable region having a random sequence or a light chain variableregion having a germ-line sequence is combined with a heavy chainvariable region in which specific amino acid residues in the heavy chainvariable region are substituted with at least one amino acid residuethat changes the antigen-binding activity according to the calcium ionconcentration. Without being particularly limited thereto, the aminoacid residues include, for example, amino acid residues of the heavychain CDR1, amino acid residues of the heavy chain CDR2, and the aminoacids at positions 95, 96, 100a, and/or 101 in the heavy chain CDR3. Aslong as the amino acid residues form a calcium-binding motif and/or theantigen-binding activity changes according to the calcium ionconcentration, the amino acid residues may be contained alone or incombination of two or more.

Even when the above-described heavy chain variable region which isintroduced with at least one amino acid residue that changes theantigen-binding activity according to the calcium ion concentration iscombined with a light chain variable region having a random sequence ora light chain variable region having a germ-line sequence, the heavychain variable region can be designed in such a way that it alsocontains flexible residues. The flexible residues are not particularlylimited in number and position, as long as the antigen-binding activityof antigen-binding domains of the present invention changes according tothe calcium ion concentration. Specifically, the CDR sequences and/or FRsequences of the heavy chain may contain one or more flexible residues.Alternatively, amino acid sequences of the CDR1, CDR2, and/or CDR3 inthe heavy chain variable region other than the amino acid residues thatchange the antigen-binding activity according to the calcium ionconcentration may be randomized sequences, as in the above-describedsynthetic libraries. Without being particularly limited, when used asthe light chain variable region, germ-line sequences preferably include,for example, those of SEQ ID NO: 58 (Vk1), SEQ ID NO: 59 (Vk2), SEQ IDNO: 60 (Vk3), and SEQ ID NO: 61 (Vk4).

In the present invention, known methods such as site-directedmutagenesis (Kunkel et al., (Proc. Natl. Acad. Sci. USA (1985) 82,488-492)) and overlap extension PCR can be appropriately employed tomodify amino acids. Furthermore, various known methods can also be usedas a method for modifying amino acids into those other than naturalamino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249;Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example,one may appropriately use a cell-free translation system (Clover Direct(Protein Express)) containing tRNAs linked with an unnatural amino acidon amber suppressor tRNAs, which are complementary to the UAG codon(amber codon) which is a stop codon.

KD values for antigen-binding domains of the present invention can bemeasured by methods known to those skilled in the art, for example,using Biacore (GE healthcare), Scatchard plot, or flow cytometer.Specifically, in the case of Biacore, antigen-binding moleculescontaining an antigen-binding domain of the present invention areimmobilized on a chip and an antigen is passed as an analyte todetermine KD. The measurement can be carried out under an acidic pHrange condition and under a neutral pH range condition to calculate thevalue of KD (acidic pH)/KD (neutral pH). Meanwhile, the measurement canbe carried out under a low calcium ion concentration condition and undera high calcium ion concentration condition to calculate the value of KD(low calcium ion concentration)/KD (high calcium ion concentration).

Antigen-binding domains of the present invention may exhibit, underdifferent types of conditions at the same time, the property that theantigen-binding activity changes according to the ion concentration. Forexample, antigen-binding domains of the present invention may have theproperty that their antigen-binding activity is lower under an acidic pHrange condition than under a neutral pH range condition, and is lowerunder a low calcium ion concentration condition than under a highcalcium ion concentration condition.

Specifically, antigens of the present invention having two or more typesof physiological activities include, for example:

activin, activin A, activin AB, activin B, activin C, activin RIA,activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB,adiponectin, aFGF, AGE, allergen, amyloid β, amyloid immunoglobulinheavy chain variable region, amyloid immunoglobulin light chain variableregion, anti-Id, antithrombin III, anthrax, apo A1, apo-serum amyloid A,apo-SAA, β-2-microglobulin, bFGF, B-lymphocyte stimulator (BLyS), BMP,BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6(Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), C10, C1 inhibitory factor, C1q,C3, C3a, C4, C5, C5a (complement 5a), cathepsin A, cathepsin B,cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L,cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CCL, CCL1/1-309,CCL11/eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2,CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1,CCL20/MIP-3-α, CCL21/SLC, CCL22/MDC, CCL23/MPIF-1, CCL24/eotaxin-2,CCL25/TECK, CCL26/eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-α,CCL3L1/LD-78-β, CCL4/MIP-1-β, CCL5/RANTES, CCL6/C10, CCL7/MCP-3,CCL8/MCP-2, CCL9/10/MTP-1-γ, Clostridium botulinum toxin, Clostridiumdifficile toxin, Clostridium perfringens toxin, connective tissue growthfactor (CTGF), CTLA-4, CX3CL1/fractalkine, CXCL, CXCL1/Gro-α, CXCL10,CXCL11/I-TAC, CXCL12/SDF-1-α/β, CXCL13/BCA-1, CXCL14/BRAK,CXCL15/lungkine, CXCL16, CXCL16, CXCL2/Gro-βCXCL3/Gro-γ, CXCL3,CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8,CXCL9/Mig, CXCL10/IP-10, DC-SIGN, digoxin, EGF like domain containingprotein 7, endotoxin, RSV F protein, F10, F11, F12, F13, F5, F9, factorIa, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, FGF,FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, fibronectin, GRO/MGSA,GRO-β, GRO-γ, Helicobacter pylori (H. pylori), hapten (NP-cap orNIP-cap), HB-EGF, HCMV gB envelope glycoprotein, Hep B gp120, Bacillusanthracis protective antigen, hepatitis C virus E2 glycoprotein,hepatitis E, hepcidin, herpes simplex virus (HSV) gB glycoprotein, HIVenvelope proteins such as GP120, HIV MIB gp120 V3 loop, HLA, HLA-DR,high mobility group box 1 (HMGB1), HSP47, Hsp90, HSV gD glycoprotein,human cytomegalovirus (HCMV), human serum albumin, human tissueplasminogen activator (t-PA), IFN-α, IFN-β, IFN-γ, IgE, IGF,immunoglobulin immune complex, immunoglobulin, influenza, inhibin,inhibin α, inhibin β, laminin 5, latency-associated peptide, latentTGF-1, latent TGF-1 bp1, LBP, LDL, leptin, Lewis-Y antigen,Lewis-Y-related antigen, LFA-1, LFA-3, lipoproteins, L-selectin, type 3nonstructural protein of hepatitis C virus (NS3), oncostatin M,osteopontin, oxidized LDL, poly glycol chains of different size (forexample, PEG-20, PEG-30, and PEG40), prekallikrein, prion protein,procalcitonin, proinsulin, prolactin, proprotein convertase PC9,prorelaxin, respiratory syncytial virus (RSV) F, rheumatoid factor, RSVFgp, Sclerostin, serum amyloid P, serum albumin, Shiga like-toxin II,syndecan-1, tenascin, TGF, TGF-α, TGF-β, TGF-β Pan Specific, TGF-β1,TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO),thyroxine binding globulin, TNF-α, TNF-β, TNIL-1, toxic metabolite,transforming growth factors (TGFs) such as TGF-α and TGF-β, VEGF, viralantigens, and von Willebrand factor (vWF). Particularly preferredexamples include HMGB1, CTGF, and IgE. These antigens are preferablyderived from mammals, particularly preferably from humans.

The gene and amino acid sequences of human HMGB1 have been depositedunder GenBank accession number NM_(—)002128 (SEQ ID NO: 22) andNP_(—)002119 (SEQ ID NO: 23), respectively. In addition to human, thegene and amino acid sequences of mouse HMGB1 have been deposited underGenBank accession number NM_(—)010439 (SEQ ID NO: 24) and NP_(—)034569(SEQ ID NO: 25), respectively; and the gene and amino acid sequences ofrat HMGB1 have been deposited under GenBank accession numberNM_(—)012963 (SEQ ID NO: 26) and NP_(—)037095 (SEQ ID NO: 27),respectively.

The gene and amino acid sequences of human CTGF have been depositedunder GenBank accession number NM_(—)001901 (SEQ ID NO: 28) andNP_(—)001892 (SEQ ID NO: 29), respectively. In addition to human, thegene and amino acid sequences of mouse CTGF have been deposited underGenBank accession number NM_(—)010217 (SEQ ID NO: 30) and NP_(—)034347(SEQ ID NO: 31), respectively; and the gene and amino acid sequences ofrat CTGF have been deposited under GenBank accession number NM_(—)022266(SEQ ID NO: 32) and NP_(—)071602 (SEQ ID NO: 33), respectively.

The gene sequences of the constant region of human IgE and mouse IgEhave been deposited under GenBank accession number L00022 (SEQ ID NO:34) and GenBank accession number X01857 (SEQ ID NO: 35), respectively.

Target molecules to which HMGB1 binds include receptor for advancedglycation endproducts (RAGE), Toll-like receptor 4 (TLR4), IL-1receptor, Toll-like receptor 2 (TLR2), thrombospondin, triggeringreceptor expressed on myeloid cells-1 (TREM-1), and CD24. Meanwhile,reported substances that enhance the binding between HMGB1 and theabove-described target molecules include DNA, RNA, lipopolysaccharide(LPS), interleukin-1β (IL-1β), chemokine (C—X—C motif) Ligand 12(CXCL12), and nucleosomes. Preferred target molecules of the presentinvention include, for example, RAGE and TLR4.

Reported target molecules to which CTGF binds include insulin-likegrowth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), integrinαvβ3, transforming growth factor-β (TGF-β), bone morphogenetic protein-4(BMP-4), LDL receptor-related protein-1 (LRP-1), vascular endothelialgrowth factor (VEGF), Wnt, heparan sulfate proteoglycans (HSPG),integrins, LDL receptor-related protein-5 (LRP-5), and LDLreceptor-related protein-6 (LRP-6).

Reported target molecules to which IgE binds include FcεRI and FcεRII.These are also preferred examples of a target molecule of the presentinvention.

The present invention provides polynucleotides encoding antigen-bindingmolecules of the present invention. Polynucleotides are primarilyconstituted with DNA, RNA, other nucleotide analogs, and such.

The present invention provides vectors carrying polynucleotides of thepresent invention. Vectors for use in the present invention are notparticularly limited in type, as long as they can stably carry insertednucleic acids, and various vectors available in the market can be used.Gene cloning vectors include, for example, M13 vectors and pUC vectors.When vectors are used to produce antigen-binding molecules of thepresent invention, expression vectors are particularly useful.Expression vectors are not particularly limited, as long as they arecapable of expressing polypeptides in vitro, in E. coli, in culturedcells, or in individual organisms. For example, vectors for in vitroexpression include pBEST vectors (Promega); vectors for expression in E.coli include pGEX, pET, and pBluescript vectors (Stratagene); vectorsfor expression in cultured cells include pME18S-FL3 vector (GenBankAccession No. AB009864); vectors for expression in animal cells includepcDNA; and vectors for expression in individual organisms include pME18Svector (Mol Cell Biol. 8: 466-472 (1988)). Polynucleotides of thepresent invention can be inserted into vectors, for example, using theIn-Fusion Advantage PCR Cloning Kit (Clontech).

The present invention provides host cells retaining vectors of thepresent invention. Host cells that can be used are not particularlylimited, and for example, E. coli and various animal cells can besuitably used. Host cells can be used, for example, as a productionsystem to produce or express antigen-binding molecules of the presentinvention. Such production systems include in vitro and in vivoproduction systems. The in vitro production systems include productionsystems using eukaryotic cells and those using prokaryotic cells.

Eukaryotic cells that can be used as host cells include, for example,animal cells, plant cells, and fungal cells. Animal cells include, forexample, mammalian cells, for example, CHO (J. Exp. Med. (1995) 108:94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, andVero; amphibian cells, for example, Xenopus oocytes (Valle et al.,Nature (1981) 291: 338-340); and insect cells, for example, Sf9, Sf21,and Tn5. CHO-DG44, CHO-DX11B, COS7, HEK293, and BHK are preferably used.CHO is particularly preferable for large-scale expression. Vectors canbe introduced into host cells, for example, by using techniques known tothose skilled in the art, such as calcium phosphate methods,DEAE-dextran methods, methods using cationic liposome DOTAP(Boehringer-Mannheim), electroporation, lipofection, and microinjection.Alternatively, the Free Style 293 Expression System (Invitrogen) may beused to accomplish the process from gene introduction to polypeptideexpression.

As plant cells, for example, cells derived from Nicotiana tabacum andLemna minor are known as a protein production system. Calluses can becultured from these cells to produce antigen-binding molecules of thepresent invention. Fungal cells known as a protein expression systeminclude yeast cells, for example, cells of genus Saccharomyces such asSaccharomyces cerevisiae and Saccharomyces pombe; and cells offilamentous fungi, for example, cells of genus Aspergillus such asAspergillus niger.

When prokaryotic cells are used, there are production systems that usebacterial cells. Bacterial cells known as a protein production systeminclude, for example, Streptococcus, Staphylococcus, Escherichia coli,streptomyces, and Bacillus subtilis.

On the other hand, for example, production systems using animals orplants may be used as systems for producing polypeptides in vivo. Apolynucleotide of interest is introduced into an animal or plant and thepolypeptide is produced in the body of the animal or plant, and thencollected. The hosts of the present invention include such animals andplants.

The production system using animals include those using mammals orinsects. It is possible to use mammals such as goats, pigs, sheep, mice,and bovines (Vicki Glaser SPECTRUM Biotechnology Applications (1993)).The mammals may be transgenic animals. For example, a polynucleotideencoding an antigen-binding molecule provided by the present inventionis prepared as a fusion gene with a gene encoding a polypeptidespecifically produced in milk, such as the goat β casein. Next, goatembryos are injected with polynucleotide fragments containing the fusiongene, and then transplanted to female goats. Desired antigen-bindingmolecules can be obtained from milk produced by the transgenic goats,which are born from the goats that received the embryos, or theiroffspring. Hormones may be administered as appropriate to increase thevolume of milk containing the antigen-binding molecule produced by thetransgenic goats (Ebert et al., Bio/Technology (1994) 12: 699-702).

Insects such as silkworms may be used to produce the antigen-bindingmolecules provided by the present invention. When silkworms are used,baculoviruses carrying a polynucleotide encoding an antigen-bindingmolecule of interest can be used to infect silkworms, and theantigen-binding molecule of interest can be obtained from their bodyfluids.

Furthermore, when plants are used to produce the antigen-bindingmolecules provided by the present invention, for example, tobacco may beused. When tobacco is used, a polynucleotide encoding an antigen-bindingmolecule of interest is inserted into a plant expression vector, forexample, pMON 530, and then the vector is introduced into bacteria, suchas Agrobacterium tumefaciens. The bacteria are then allowed to infecttobacco such as Nicotiana tabacum, and the desired antigen-bindingmolecules can be collected from their leaves (Ma et al., Eur. J.Immunol. (1994) 24: 131-138). Alternatively, it is possible to infectduckweed (Lemna minor) with similar bacteria. After cloning, the desiredantigen-binding molecules can be obtained from the duckweed cells (Cox KM et al., Nat. Biotechnol. 2006 December; 24(12): 1591-1597).

The thus obtained antigen-binding molecules may be isolated from theinside or outside (such as the medium and milk) of host cells, andpurified as substantially pure and homogenous molecules. The methods forisolating and purifying antigen-binding molecules provided by thepresent invention are not particularly limited, and isolation andpurification methods usually used for polypeptide purification can beused. Isolation and purification may be performed by appropriatelyselecting and combining, for example, column chromatographies,filtration, ultrafiltration, salting out, solvent precipitation, solventextraction, distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, and recrystallization.

Chromatography includes, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse-phase chromatography, and adsorptionchromatography (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., (1996) Cold Spring Harbor Laboratory Press). Such chromatographicmethods can be conducted using liquid phase chromatography such as HPLCand FPLC. Columns used for affinity chromatography include, protein Acolumns and protein G columns. Columns using protein A include, forexample, Hyper D, POROS, and Sepharose F. F. (Pharmacia).

If needed, an antigen-binding molecule provided by the present inventioncan be modified arbitrarily, and peptides can be partially deleted byallowing an appropriate protein modification enzyme to act on theantigen-binding molecule. Such protein modification enzymes include, forexample, trypsin, chymotrypsin, lysyl endopeptidases, protein kinases,and glucosidases.

The present invention also provides pharmaceutical compositionscomprising an antigen-binding molecule of the present invention as anactive ingredient. Pharmaceutical compositions can be used to treatdiseases. It is preferable that pharmaceutical compositions of thepresent invention are used to treat diseases for which one of the causesis assumed to be an antigen that has physiological activity. The antigenis preferably an antigen having two or more types of physiologicalactivities that can be reduced in vivo by antigen-binding molecules ofthe present invention. In the present specification, “treatment” meansto obtain pharmacological and/or physiological effects. Such an effectmay be preventive in the sense that it completely or partially preventsthe symptoms of a disease, or may be therapeutic in the sense that itcompletely or partially cures the symptoms of a disease. In the presentspecification, “treatment” includes all of the treatments for diseasesin mammals, in particular humans. Furthermore, the “treatment” alsoincludes preventing the onset of diseases in subjects who have not yetbeen diagnosed with a disease, restraining the progression of symptoms,and reducing the symptoms of diseases.

Pharmaceutical compositions of the present invention can be formulatedby methods known to those skilled in the art (for example, Remington'sPharmaceutical Science, latest edition, Mark Publishing Company, Easton,USA). If needed, antigen-binding molecules of the present invention maybe formulated in combination with other pharmaceutical ingredients. Thepharmaceutical compositions may also comprise, for example,pharmaceutically acceptable carriers and additives. The pharmaceuticalcompositions of the present invention can also be used parenterally, forexample, when they are formulated in a sterile solution or suspensionfor injection using water or any other pharmaceutically acceptableliquid. Dosage forms for oral and parenteral administration, and methodsfor producing them are well known to those skilled in the art, and maybe produced according to conventional methods by mixing thepharmaceutical compositions of the present invention withpharmaceutically acceptable carriers and such. In the present invention,examples include sterile water, physiological saline, vegetable oils,emulsifiers, surfactants, excipients, vehicles, colorants, flavoringagents, preservatives, antiseptic agents, stabilizers, buffers,suspension agents, isotonizing agents, binders, disintegrating agents,lubricants, fluidity enhancing agents, flavor additives, and corrigents;however, the carriers are not limited to the above example, and otherconventional carriers may be appropriately used. Specifically, lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose,hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate,polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, saccharose,carboxymethylcellulose, corn starch, and inorganic salts may be used.Pharmaceutical compositions of the present invention may be formulatedby appropriately combining the above-described examples, and mixing theminto unit dosage forms required for generally accepted drug manufacture.The amount of active ingredients in these preparations is determined toachieve an adequate dose within the indicated range.

Pharmaceutical compositions of the present invention can be administeredorally or parenterally; however, parenteral administration is preferred,which specifically includes injection, transnasal administration,transpulmonary administration, and transdermal administration. Injectionincludes, for example, intravenous administration, intramuscularadministration, intraperitoneal administration, and subcutaneousadministration. The dose can be suitably selected from the range of0.0001 mg to 1000 mg/kg body weight of a patient or within the range of0.001 mg to 10000 mg/patient; however, the dose is not limited to thisexample. Subjects to be administered are mammals, preferably humans.

The present invention also provides kits comprising an antigen-bindingmolecule of the present invention or a pharmaceutical composition of thepresent invention, and kits for use in various methods of the presentinvention. The kits of the present invention may additionally contain ina package instruction manuals describing how to use them, as necessary.In addition, the kits of the present invention can be preferably usedin: (i) methods for reducing antigen concentration in plasma; (ii)methods for enhancing antigen incorporation into cells; or (iii) methodsfor reducing the physiological activity of antigens in vivo.

In the present invention, preferred antigens include, for example,HMGB1. Diseases for which one of the causes is assumed to be HMGB1include sepsis, trauma, acute respiratory distress syndrome (ARDS),ischemia-reperfusion injury in the brain, heart, liver, kidney, andsuch, pancreatitis, nephritis, hepatitis, colitis, meningitis,endophthalmitis, myopathy, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE), diabetes, multiple sclerosis (MS), colorectalcancer, osteosarcoma, cervical cancer, liver cancer, lymphoma,nasopharyngeal cancer, prostate cancer, skin cancer, urothelial cancer,lung cancer, autism, seizure, sleep apnea syndrome, HIV infection,pulmonary fibrosis, and burn injury. Other preferred antigens include,for example, CTGF. Diseases for which one of the causes is assumed to beCTGF include fibrosis such as pulmonary fibrosis and hepatic fibrosis.Other preferred antigens include, for example, IgE. Diseases for whichone of the causes is assumed to be IgE include allergic diseases such asbronchial asthma, atopic dermatitis, and pollinosis.

The present invention also provides methods for producingantigen-binding molecules of the present invention, which comprise thesteps of:

-   -   (a) selecting an antigen having two or more types of        physiological activities;    -   (b) obtaining an antigen-binding domain;    -   (c) obtaining at least one receptor-binding domain;    -   (d) selecting from antigen-binding domains obtained in step (b)        a domain of which antigen-binding activity changes according to        the ion concentration;    -   (e) selecting from receptor-binding domains obtained in step (c)        a domain that has human FcRn-binding activity under an acidic pH        range condition and of which human Fc receptor-binding activity        under a neutral pH range condition is greater than the human Fc        receptor-binding activity of native human IgG;    -   (f) producing an antigen-binding molecule in which the        antigen-binding domain selected in step (d) is linked to the        receptor-binding domain selected in step (e); and    -   (g) selecting from antigen-binding molecules produced in        step (f) an antigen-binding molecule which inhibits one or more        types of the physiological activities of the antigen by binding        to the antigen while allowing for the antigen to retain at least        one type of physiological activity.

Antigen-binding domains of the present invention may be prepared by anymethods. For example, when an antigen-binding domain is an antibody or afragment thereof (such as a variable region, Fab, F(ab′)2, Fv, or CDR),it can be prepared by antibody library methods, hybridoma methods, Bcell cloning methods (Bernasconi et al., Science (2002) 298, 2199-2202;or WO2008/081008), and such. Meanwhile, antibodies and fragments thereofprepared as described above may be modified at at least one amino acid.

There are a number of known methods for constructing antibody libraries.Regarding phage display libraries, for example, those skilled in the artcan prepare them by referring to documents such as Clackson et al.,Nature 1991, 352: 624-8; Marks et al., J. Mol. Biol. 1991, 222: 581-97;Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6; Griffiths etal., EMBO J. 1994, 13: 324.0-60; Vaughan et al., Nature Biotechnology1996, 14: 309-14; Kang A S et al., Proc Natl Acad Sci USA (1991) 88,4363-4366; and Japanese Patent Kohyo Publication No. (JP-A) H10-504970(unexamined Japanese national phase publication corresponding to anon-Japanese international publication). When the variable regions ofantibodies in a phage display library are expressed as single-chainantibodies (scFv) on the surface of phages, phages that bind to antigenscan be selected by panning. The genes of selected phages are analyzed todetermine the DNA sequences encoding the variable regions of antibodiesthat bind to the antigens. Once the DNA sequences of the variableregions of antibodies are revealed, they can be linked to the DNAsencoding desired constant regions and inserted into appropriateexpression vectors. The resulting vectors are introduced into host cellsfor expression to produce the antibodies as recombinant proteins. Thesemethods are already well known, and one can refer to the followingdocuments: WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172,WO95/01438, and WO95/15388. In addition to phage display libraries, itis possible to use known antibody libraries such as ribosome displaylibraries (Schaffitzel C et al., J. Immunol. Methods (1999) 231,119-135.), cell display libraries (Fuchs P et al., Biotechnology (1991)9, 1369-1372; Boder E T & Wittrup K D, Nat Biotechnol (1997) 15,553-557; WO95/15393), nucleotide display libraries (Cull M G et al.,Proc Nalt Acad Sci USA (1992) 89, 1865-1869; Roberts R D & Szostak J W,Proc Natl Acad Sci USA (1997) 94, 12297-12302), and eukaryotic virusdisplay libraries (Grabherr R & Ernst W, Comb Chem High ThroughputScreen (1991) 4, 185-192).

As a hybridoma method for preparing antibodies, basically knowntechniques are used. A desired antigen or cells expressing the desiredantigen are used as a sensitizing antigen. Immune cells obtained byimmunization with the antigen according to a conventional immunizationmethod are fused with known parental cells by a conventional cell fusionmethod. The resulting cells are screened for cells producing monoclonalantibodies (hybridomas) by a conventional screening method. Furthermore,by using reverse transcriptase, cDNAs encoding the variable regions ofthe antibodies can be prepared from the mRNA of the obtained hybridomas.The cDNAs are linked to the DNA encoding a desired constant region andinserted into an appropriate expression vector. The resulting vectorsare introduced and expressed in host cells to produce the antibodies asrecombinant proteins.

More specifically, the sensitizing antigens include, but are not limitedto, for example, both complete antigens with immunogenicity, andincomplete antigens without immunogenicity including haptens. Forexample, the full-length proteins of interest and partial peptides canbe used. In addition, it is known that substances composed ofpolysaccharides, nucleic acids, lipids, and such can serve as anantigen, and are not particularly limited. Antigens can be prepared bymethods known to those skilled in the art, for example, according tomethods using baculoviruses (for example, WO98/46777). Hybridomas can beproduced, for example, according to the method of Milstein et al. (G.Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). When theimmunogenicity of an antigen is low, it can be linked to a macromoleculehaving immunogenicity, such as albumin, and then used for immunization.Alternatively, the antigen may be linked to other molecules, ifnecessary.

Hybridomas can be obtained by immunizing animals using suitablesensitizing antigens described above. Alternatively, antibody-producingcells can be prepared by in vitro immortalization of lymphocytes capableof producing antibodies. Various mammals can be used for immunization,and such animals generally used include rodents, lagomorphas andprimates. Such animals include, for example, rodents such as mice, rats,and hamsters; lagomorphas such as rabbits; and primates such as monkeysincluding cynomolgus monkeys, rhesus monkeys, hamadryas baboons, andchimpanzees. In addition, transgenic animals with the repertoire ofhuman antibody genes are also known, and such animals can be immunizedwith a desired antigen to obtain human antibodies (see WO93/12227;WO92/03918; WO94/02602; WO96/34096; WO96/33735; Mendez et al., Nat.Genet. 1997, 15: 146-56). Instead of using such transgenic animals, forexample, human lymphocytes can be sensitized in vitro with a desiredantigen, and the sensitized lymphocytes can be fused with human myelomacells, for example, U266 to obtain desired human antibodies withantigen-binding activity (see Japanese Patent Kohyo Publication No.(JP-A) H01-59878 (unexamined Japanese national phase publicationcorresponding to a non-Japanese international publication)).

Animals are immunized, for example, by appropriately diluting andsuspending a sensitizing antigen in phosphate-buffered saline (PBS),physiological saline, or such, and after combining it with an adjuvantand emulsifying if needed, injecting it intraperitoneally orsubcutaneously to the animals. Then, the sensitizing antigen combinedwith Freund's incomplete adjuvant is preferably administered severaltimes every four to 21 days. Antibody production can be confirmed bymeasuring the titer of antibody of interest in the sera of the animalsaccording to conventional methods.

To produce hybridomas, antibody-producing cells such as lymphocytesobtained from animals immunized with a desired antigen are fused withmyeloma cells using conventional fusion agents (for example,polyethylene glycol) (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, 1986, 59-103). If needed, hybridomas can becultured and grown, and the binding specificity of antibodies producedby the hybridomas can be measured using known analysis methods, such asimmunoprecipitation, radioimmunoassay (RIA), and enzyme-linkedimmunosorbent assay (ELISA). Then, as necessary, hybridomas producingthe antibodies of interest with determined specificity, affinity, oractivity can be subcloned by methods such as limiting dilution.

Next, genes encoding antibodies can be cloned from hybridomas orantibody-producing cells (such as sensitized lymphocytes) using probescapable of specifically binding to antibody genes (for example,oligonucleotides complementary to sequences encoding the antibodyconstant regions). Alternatively, the genes can be cloned from mRNA byRT-PCR. Immunoglobulins are classified into five different classes: IgA,IgD, IgE, IgG, and IgM. These classes are further divided into severalsubclasses (isotypes) (for example, IgG1, IgG2, IgG3, and IgG4).

Receptor-binding domains of the present invention may be obtained by anymethods. For example, when a receptor-binding domain is an anti-FcRnantibody or a fragment thereof (a variable region, Fab, F(ab′)2, Fv,CDR, etc.), it can be prepared by the above-described antibody librarymethods and hybridoma methods. Other examples of receptor-bindingdomains include the Fc region of an antibody (IgG) and regionscontaining the Fc region (antibody constant regions and full lengthantibodies). When a receptor-binding domain is the Fc region of an IgG,it may be modified at at least one amino acid.

Arbitrary amino acids can be added, deleted, and/or substituted inpolypeptides by methods known to those skilled in the art, for example,site-directed mutagenesis (Hashimoto-Gotoh T. et al., Gene (1995) 152,271-275; Zoller M. J. & Smith M., Methods Enzymol (1983) 100, 468-500;Kramer W. et al., Nucleic Acids Res (1987) 12, 9441-9456; Kramer W. &Fritz H. J., Methods Enzymol (1987) 154, 350-367; Kunkel T. A., ProcNatl Acad Sci USA (1985) 82, 488-492).

Methods for producing chimeric antibodies are known, and in the case ofa human-mouse chimeric antibody, for example, DNA encoding the variableregion of a mouse antibody can be linked to a DNA encoding the constantregion of a human antibody and inserted into an expression vector, andthen introducing the vector into a host to produce the chimericantibody.

Humanized antibodies are also referred to as “reshaped humanantibodies”, and result from grafting the complementarity determiningregion (CDR) of an antibody derived from a nonhuman mammal, for example,a mouse, into a human antibody at its CDR. Methods for identifying CDRsare known (Kabat et al., Sequence of Proteins of Immunological Interest(1987) National Institute of Health, Bethesda, Md.; Chothia et al.,Nature (1989) 342: 877). General genetic recombination techniques forgrafting CDRs are also known (see European Patent ApplicationPublication No. EP 125023; WO96/02576). Humanized antibodies can beproduced by known methods, for example, with a system using commonexpression vectors, by determining the CDR of a mouse antibody andpreparing a DNA encoding an antibody in which the CDR is linked to theframework region (FR) of a human antibody. Such DNAs can be synthesizedby assemble PCR, using as primers several oligonucleotides designed toinclude overlapping portions at the ends of both the CDR and FR regions(see the method described in WO98/13388). Human antibody FRs linked viaCDRs are selected such that the CDRs form a suitable antigen bindingsite. If required, amino acids in the FRs of an antibody variable regionmay be altered in such a way that the CDRs of the reshaped humanantibody form a suitable antigen binding site (Sato et al., Cancer Res.(1993) 53: 10.01-6). Amino acid residues that can be altered in the FRsinclude portions that directly bind to an antigen via non-covalent bonds(Amit et al., Science (1986) 233: 747-53), portions that influence oract on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196:901-17), and portions involved in the interaction between VH and VL (EP239400).

In addition to the humanization techniques described above, antibodiesmay be modified to improve their biological properties, for example,antigen affinity. In the present invention, alteration can be achievedusing methods such as site-directed mutagenesis (see, for example,Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis,and cassette mutagenesis. In general, mutant antibodies with improvedbiological properties show amino acid sequence homology and/orsimilarity of 70% or higher, more preferably 80% or higher, and evenmore preferably 90% or higher (for example, 95% or higher, 96%, 97%,98%, 99%, etc.), to the amino acid sequence of the original antibodyvariable region. In the present specification, sequence homology and/orsimilarity is defined as the ratio of amino acid residues that arehomologous (same residue) and/or similar (residues classified into thesame group based on the general properties of amino acid side chains) tothe amino acid residues of the original sequence, after the sequencehomology value has been maximized by sequence alignment and gapintroduction, as necessary. In general, native amino acid residues areclassified based on the characteristics of their side chains into thefollowing groups:

-   -   (1) hydrophobic: alanine, isoleucine, valine, methionine, and        leucine;    -   (2) neutral, hydrophilic: asparagine, glutamine, cysteine,        threonine, and serine;    -   (3) acidic: aspartic acid and glutamic acid;    -   (4) basic: arginine, histidine, and lysine;    -   (5) residues that have influence on chain configuration: glycine        and proline; and    -   (6) aromatic: tyrosine, tryptophan, and phenylalanine.

In methods for producing antigen-binding molecules of the presentinvention, a number of antigen-binding domains are prepared, and domainsof which antigen-binding activity changes according to the ionconcentration can be selected from among them; or a number of domainsare prepared by modifying an arbitrary antigen-binding domain by adding,deleting, and/or substituting at least one amino acid, and domains ofwhich antigen-binding activity changes according to the ionconcentration can be selected from among them. Alternatively,antigen-binding domains that inhibit one or more of the physiologicalactivities of an antigen by binding to the antigen but allow for theantigen to retain at least one type of physiological activity may beappropriately selected from a number of antigen-binding domains.

In methods for producing antigen-binding molecules of the presentinvention, whether the binding of the antigen-binding molecule or theantigen-binding domain thereof to an antigen inhibits one or more typesof the physiological activities of the antigen or allows for the antigento retain at least one type of physiological activity can also beconfirmed, by measuring whether one or more of the activities of theantigen to bind target molecules are inhibited or whether the activityto bind at least one type of target molecule is retained.

In methods for producing antigen-binding molecules of the presentinvention, a number of receptor-binding domains are prepared, and fromamong them, domains that have human FcRn-binding activity under anacidic pH range condition, and of which human Fc receptor-bindingactivity under a neutral pH range condition is greater than that ofnative human IgG can be selected; or a number of domains are prepared bymodifying an arbitrary receptor-binding domain by adding, deleting,and/or substituting at least one amino acid, and from among them,domains that have human FcRn-binding activity under an acidic pH rangecondition, and of which human Fc receptor-binding activity under aneutral pH range condition is greater than that of native human IgG canbe selected.

In methods for producing antigen-binding molecules of the presentinvention, in order to produce an antigen-binding molecule in which anantigen-binding domain is linked to a receptor-binding domain,polynucleotides encoding the antigen-binding domain and thereceptor-binding domain are constructed, linked together in frame,inserted into an expression vector, and expressed in host cells. Theantigen-binding domain may be linked directly to the receptor-bindingdomain, or indirectly using an arbitrary peptide linker that can beintroduced by genetic engineering or using a synthetic compound linker(for example, the linkers disclosed in Protein Engineering (1996) 9,299-305). Such peptide linkers are not particularly limited in lengthand amino acid sequence; however, peptides of 100 amino acids or less,preferably 50 amino acids or less, more preferably 30 amino acids orless, and particularly preferably 10 amino acids or less are generallyused.

The present invention also provides methods of screening for antibodiesof which antigen-binding activity changes depending on conditions, whichcomprise the steps of:

-   -   (a) preparing antibody-producing cells;    -   (b) contacting an antigen with the cells of (a) under the first        condition;    -   (c) selecting from the cells of step (b), cells bound to a        specific amount of antigen or more;    -   (d) exposing the cells of step (c) to the second condition; and    -   (e) selecting from the cells of step (d), cells in which the        amount of antigen binding has been reduced as compared to step        (c).    -   More preferred embodiments of the above-described method include        methods comprising the steps of:    -   (a) preparing antibody-producing cells;    -   (b) contacting an antigen with the cells of (a) under the first        condition;    -   (c) contacting an anti-IgG antibody with the cells of step (b);    -   (d) selecting from the cells of step (c), cells bound to a        specific amount of antigen or more and bound to a specific        amount of anti-IgG antibody or more;    -   (e) exposing the cells of step (d) to the second condition; and    -   (f) selecting from the cells of step (e), cells in which the        amount of antigen binding has been reduced as compared to step        (d).

Still more preferred embodiments of the above-described method includemethods comprising the steps of:

-   -   (a) preparing antibody-producing cells;    -   (b) contacting an antigen with the cell of (a) under the first        condition;    -   (c) enriching cells bound to the antigen among the cells of step        (b);    -   (d) contacting an anti-IgG antibody with the cells of step (c);    -   (e) selecting from the cells of step (d), cells bound to a        specific amount of antigen or more and bound to a specific        amount of anti-IgG antibody or more;    -   (f) exposing the cells of step (e) to the second condition; and    -   (g) selecting from the cells of step (f), cells in which the        amount of antigen binding has been reduced as compared to step        (e).

In the present invention, “antibody-producing cells” are notparticularly limited as long as they contain an antibody gene andexpress the antibody protein; however, they are preferablynaturally-occurring cells producing antibodies within an animal body,more preferably lymphocytes, and still more preferably B cells. Animalscan be appropriately selected from various mammals (such as mice, rats,hamsters, rabbits, cynomolgus monkeys, rhesus monkeys, hamadryasbaboons, chimpanzees, and humans); however, rabbits are particularlypreferable in the present invention. It is also preferable to useanimals immunized with desired antigens. Antibody-producing cells may benaturally-occurring cells or artificially produced cells such ashybridomas and genetically modified cells. Antibody-producing cells foruse in the present invention preferably have the property that theysecrete antibodies to the outside of the cells (secretory antibodies)and/or the property that they present antibodies on the cell membrane(membrane-bound antibodies). Naturally occurring antibody-producingcells in the animal body can be preferably collected, for example, fromspleen, lymph nodes, and blood (peripheral blood mononuclear cells).Such methods are known to those skilled in the art, and are alsodescribed in the Examples below.

In the present invention, “the first condition” and “the secondcondition” mean two types of different conditions and arbitraryconditions can be set when one desires to obtain an antibody of whichantigen-binding activity varies between the conditions. Preferredexamples in the present invention include extracellular condition andintracellular condition. The category of conditions is not particularlylimited as long as antibody-producing cells are exposed to theconditions, and examples include temperature, pH, compositions in media,and the concentrations thereof. The conditions preferably include ionconcentration, particularly preferably hydrogen ion concentration (pH)and calcium ion concentration. The intracellular condition preferablyrefers to a condition characteristic of the internal environment of anendosome, and the extracellular condition preferably refers to acondition characteristic of the environment in plasma.

The extracellular pH is neutral as compared to that inside the cell, andconversely the intracellular pH is acidic as compared to that outsidethe cell. A neutral pH range preferred in the present invention is pH6.7 to pH 10.0, more preferably pH 7.0 to pH 9.0, still more preferablyany of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, andparticularly preferably pH 7.4 which is close to the pH in plasma (inblood). Meanwhile, an acidic pH range preferred in the present inventionis pH 4.0 to pH 6.5, more preferably pH 5.0 to pH 6.5, still morepreferably any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,and 6.5, and particularly preferably pH 5.8 to pH 6.0 which is close tothe pH in the early endosome in vivo.

Meanwhile, the extracellular calcium ion concentration is higher thanthat inside the cell, and conversely the intracellular calcium ionconcentration is lower than that outside the cell. A high calcium ionconcentration preferred in the present invention is 100 μM to 10 mM,more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to2.5 mM which is close to the calcium ion concentration in plasma (inblood). Meanwhile, a low calcium ion concentration preferred in thepresent invention is 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM,and particularly preferably 1 μM to 5 μM which is close to the calciumion concentration in the early endosome in vivo. Low calcium ionconcentration can also be achieved by adding a chelating agent such asEDTA, instead of reducing the amount of calcium added.

“The first condition” and “the second condition” may include severalconditions at the same time. For example, “the first condition” may be aneutral pH condition and a high calcium ion concentration condition, and“the second condition” may be an acidic pH condition and a low calciumion concentration condition.

Antigens may be any substances as long as it is possible to produceantibodies against them. The type of antigen is not particularlylimited, and it preferably comprises polypeptides. Meanwhile, in theabove-described antibody screening methods of the present invention,antigens are preferably labeled with certain substances that can bedetected with high sensitivity. The labeling substances may be linkeddirectly to antigens, or indirectly to antigens using antigen-antibodyreaction or biotin-avidin reaction. The labeling substances include, forexample, radioisotopes, chemiluminescent compounds, fluorescentcompounds, phosphorescent compounds, magnetic particles, and enzymes,particularly preferably fluorescent compounds. Fluorescent compoundsinclude, for example, fluorescein isothiocyanate (FITC), phycoerythrin(PE), PE-Cyanin5 (PE-Cy5), PE-Cyanin5.5 (PE-Cy5.5), PE-Cyanin7 (PE-Cy5),rhodamine isothiocyanate, Texas Red, PE-Texas Red-x (ECD),allophycocyanin (APC), APC-Cyanin 7 (APC-Cy7, PharRed), PeridininChlorophyll Protein (Per-CP), and Per-CP-Cyanin5.5 (PerCP-Cy5.5).

Furthermore, in the above-described antibody screening methods of thepresent invention, anti-IgG antibodies are also preferably labeled withcertain substances that can be detected with high sensitivity. Cells towhich anti-IgG antibodies are bound can be selected fromantibody-producing cells to increase the percentage of cells expressingIgG subclasses. The presence of IgG expression in B cells namelysuggests that class switching to IgG is taking place. To enrich cellsproducing such matured antibodies is expected to be beneficial from theviewpoint of screening for antibodies with strong binding activity. Thelabeling substances may be linked directly to antigens, or indirectly toantigens using antigen-antibody reaction or biotin-avidin reaction. Thelabeling substances include, for example, those described above. When anantigen and an anti-IgG antibody are both used, it is desirable that thelabeling substances for them are different from each other and theirdetection methods are different (each can be detected independently).Antigens and anti-IgG antibodies can be labeled by referring to methodsknown to those skilled in the art (for example, U.S. Pat. No. 5,057,313and U.S. Pat. No. 5,156,840).

In the above-described antibody screening methods provided by thepresent invention, the step of selecting cells bound to a specificamount of antigen or more and/or a specific amount of anti-IgG antibodyor more is preferably achieved by detecting the above-described labelingsubstances. For example, when an antigen and/or an anti-IgG antibody areeach labeled with a different type of fluorescent compound, whether aspecific amount of antigen or more and/or a specific amount of anti-IgGantibody or more is bound to cells can be assessed by testing whetherthe fluorescence emitted from each fluorescent compound is detected tobe at a specific lever or higher. The specific level can be setarbitrarily by those skilled in the art depending on the purpose. In thepresent invention, it is preferable that the selection step is achievedusing FACS (Fluorescence Activated Cell Sorting).

In the above-described antibody screening methods provided by thepresent invention, it is preferable that the step of enrichingantigen-bound cells is achieved by detecting the above-describedlabeling substances. For example, when an antigen is labeled withmagnetic particles, antigen-bound cells can be separated from cells notbound by the antigen using magnetic devices. Antigen-bound cells can beenriched by removing cells not bound by the antigen. In the presentinvention, it is preferable that the enriching step is achieved by usingthe MACS (Magnetic Activated Cell Sorting (Registered Trademark)).

By using the above-described antibody screening methods provided by thepresent invention, a large amount of antibody-producing cells can besimply and efficiently screened for antibodies with antigen-bindingactivity that changes according to condition. As compared toconventional methods, the methods enable one to drastically increase thenumber of cells that can be screened, and thus greatly raise theprobability to find rare antibodies that have been previouslyundetectable. Thus, the above-described antibody screening methods ofthe present invention are useful.

The present invention also provides methods for reducing antigenconcentration in plasma by administering an antigen-binding molecule ofthe present invention, methods for enhancing antigen incorporation intocells by administering an antigen-binding molecule of the presentinvention, and methods for reducing the physiological activity ofantigens in vivo by administering an antigen-binding molecule of thepresent invention.

The present invention also provides therapeutic agents for diseases,which comprise an antigen-binding molecule of the present invention asan active ingredient. The diseases include, for example, diseases forwhich one of the causes is assumed to be HMGB1, diseases for which oneof the causes is assumed to be CTGF, and diseases for which one of thecauses is assumed to be IgE.

The present invention also provides kits comprising antigen-bindingmolecules of the present invention for use in methods for reducingantigen concentration in plasma, methods for enhancing antigenincorporation into cells, and methods for reducing the physiologicalactivity of antigens in vivo.

The present invention also provides methods for treating diseases forwhich one of the causes is assumed to be an antigen that hasphysiological activity, methods for reducing antigen concentration inplasma, methods for enhancing antigen incorporation into cells, andmethods for reducing the physiological activity of antigens in vivo,which comprise the step of administering antigen-binding molecules ofthe present invention.

The present invention also provides agents for treating diseases forwhich one of the causes is assumed to be an antigen that hasphysiological activity, agents for reducing antigen concentration inplasma, agents for enhancing antigen incorporation into cells, andagents for reducing the physiological activity of antigens in vivo,which comprise an antigen-binding molecule of the present invention asan active ingredient.

The present invention also provides antigen-binding molecules of thepresent invention for use in methods for treating diseases for which oneof the causes is assumed to be an antigen that has physiologicalactivity, methods for reducing antigen concentration in plasma, methodsfor enhancing antigen incorporation into cells, and methods for reducingthe physiological activity of antigens in vivo.

The present invention also provides the use of antigen-binding moleculesof the present invention in the production of agents for treatingdiseases for which one of the causes is assumed to be an antigen thathas physiological activity, agents for reducing antigen concentration inplasma, agents for enhancing antigen incorporation into cells, andagents for reducing the physiological activity of antigens in vivo.

The present invention also provides processes for producing agents fortreating diseases for which one of the causes is assumed to be anantigen that has physiological activity, agents for reducing antigenconcentration in plasma, agents for enhancing antigen incorporation intocells, and agents for reducing the physiological activity of antigens invivo, which comprise the step of using an antigen-binding molecule ofthe present invention. Such diseases include, for example, diseases forwhich one of the causes is assumed to be HMGB1, diseases for which oneof the causes is assumed to be CTGF, and diseases for which one of thecauses is assumed to be IgE.

Amino acids contained in the amino acid sequences of the presentinvention may be post-translationally modified (for example, themodification of an N-terminal glutamine into a pyroglutamic acid bypyroglutamylation is well-known to those skilled in the art). Naturally,such post-translationally modified amino acids are included in the aminoacid sequences in the present invention.

All prior art documents cited in the specification are incorporatedherein by reference.

EXAMPLES

Herein below, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1 Construction of Anti-HMGB1 Antibodies by the Rabbit B CellCloning Method Preparation of HMGB1

HMGB1 was prepared as an antigen by the following procedure. An animalcell expression vector inserted with a DNA sequence encoding human HMGB1(GenBank accession number NP002119, SEQ ID NO: 7) was constructed andused in combination with FreeStyle293 (Invitrogen) to express thefull-length human HMGB1 protein in the culture supernatant. From theresulting culture supernatant, the HMGB1 protein was purified bycation-exchange column chromatography, anion-exchange chromatography,and gel filtration chromatography.

Immunization of Animals with the Antigen

Rabbits were immunized with HMGB1. The initial immunization was carriedout by intracutaneously injecting 100 μg of the HMGB1 protein includedin complete Freund's adjuvant (CFA). Then, booster immunization wasperformed with the HMGB1 protein included in incomplete Freund'sadjuvant (IFA) at 50 μg each time, twice or more times at intervals ofone week or more. Antibody titers were determined to confirm antibodyproduction in the animal bodies.

Tissue Sampling from Immunized Animals and Preparation of Single-CellSuspensions

Animals which had been confirmed to produce antibodies were euthanizedto collect their spleens, lymph nodes, and blood. Peripheral bloodmononuclear cells (PBMCs) were prepared from the blood. An equal volumeof the blood was carefully overlaid onto Histpaque-1077 (Sigma) in a50-ml centrifuge tube, and centrifuged at 400×g and 25° C. for 30minutes. After centrifugation, the PBMC layer was carefully collectedwith a glass Pasteur pipette and transferred into a sterile 50-ml tube.About 10 volumes of RPMI-1640 containing 2% FBS was added to thecollected cell suspension. The cells were washed by centrifugation at1000×g for 5 minutes followed by removing the supernatant. The samewashing treatment was carried out again to prepare PBMCs. The PBMCs werestained with trypan blue, and the cell density was determined with ahemocytometer.

The collected spleens and lymph nodes were filtered through 70 μm CellStrainers (BD Falcon) using the plunger of a 5-ml syringe to preparesingle-cell suspensions. The cells were collected into sterile 50-mltubes using RPMI medium containing 2% FBS. The cells were washed bycentrifugation at 1000×g for 5 minutes followed by removing thesupernatant. 50 ml of RPMI-1640 containing 2% FBS was added, and thecells were washed again. After the final washing, the cells were stainedwith trypan blue, and the cell density was determined with ahemocytometer.

Collection of Antigen-Binding B Cells

The single-cell suspensions from the blood, spleens, and lymph nodesprepared by the above method were centrifuged at 1000×g for 5 minutestwice to wash the cells with HBSS (20 mM HEPES, 5.3 mM KCl, 0.4 mMKH₂PO₄, 4.2 mM NaHCO₃, 0.3 mM Na₂HPO₄, 0.1% BSA, 2 mM CaCl₂, 5 mMglucose, 138 mM NaCl, pH 7.4). A solution with biotinylated human HMGB1diluted to 500 nM with HBSS was prepared and added to the cells so thatthe density is 1E08 cells/100 μl or less, and the cells were suspended.Biotinylated HMGB1 was prepared by labeling the HMGB1 protein usingEZ-Link NHS-PEG4-Biotin and Biotinylation Kit (Thermo Scientific)according to the attached protocol, and then dialyzing against TBS/300mM NaCl (10 mM Tris-HCl/300 mM NaCl) using a Microdialyzer (TOMY). Thecell suspensions were incubated on ice for 30 minutes. Then, the cellswere washed with 50 ml of HBSS to remove biotinylated HMGB1 that was notbound to the cells. A solution of MACS (Registered Trademark)streptavidin beads (Miltenyi Biotech) diluted 10 times with HBSS wasprepared and added to the cells so that the density was 1E08 cells/500μl or less to suspend them. The cell suspensions were incubated on icefor 30 minutes. After washing with 50 ml of HBSS, the cells werecombined with HBSS so that the density was 1E08 cells/500 μl or less,and suspended. From the cell suspensions, fractions of positive cells towhich MACS streptavidin beads were bound were collected using theautoMACS Pro Separator.

A solution of biotinylated HMGB1 diluted with HBSS was prepared again,and added to the collected cells so that the density was 1E08 cells/100μl or less, and the cells were suspended. After incubating the cellsuspensions on ice for 30 minutes, the cells were washed with 50 ml ofHBSS. This secondary incubation with biotinylated human HMGB1 wasomitted in some cases. Solutions of streptavidin-FITC (BD) and mouseanti-rabbit IgG-PE (Southern Biotech) diluted with HBSS were preparedand added to the cells so that the density was 1E08 cells/100 μl orless, and the cells were suspended. After incubating the cellsuspensions on ice for 30 minutes, the cells were washed with 50 ml ofHBSS. Next, HBSS was added to the cells so that the density was 1E07cells/100 μl or less and they were suspended. The cell fractions whoseFITC and PE fluorescence intensities were both high were collected fromthe cell suspensions using FACSAria (BD).

Collection of B Cells that Express Antigen-Binding Antibodies WhoseDissociation Ability is Altered Depending on the Change in pH or Ca²⁺Ion Concentration

B cells that express antibodies whose dissociation ability is altereddepending on the change in pH or Ca²⁺ ion concentration were enrichedand collected by the following method. The prepared single-cellsuspensions from blood, spleens, and lymph nodes were centrifuged at1000×g for 5 minutes twice to wash the cells with HBSS (20 mM HEPES, 5.3mM KCl, 0.4 mM KH₂PO₄, 4.2 mM NaHCO₃, 0.3 mM Na₂HPO₄, 0.1% BSA, 2 mMCaCl₂, 5 mM glucose, 138 mM NaCl, pH 7.4). A solution of biotinylatedHMGB1 diluted to 500 nM with HBSS was prepared and added to the cells sothat the density was 1E08 cells/100 μl or less, and the cells weresuspended. After incubating the cell suspensions on ice for 30 minutes,the cells were washed with 50 ml of HBSS to remove biotinylated HMGB1that was not bound to the cells. A solution of MACS streptavidin beads(Miltenyi Biotech) diluted 10 times with HBSS was prepared and added tothe cells so that the density was 1E08 cells/500 μl or less, and thecells were suspended. The cell suspensions were incubated on ice for 30minutes. After washing with 50 ml of HBSS, HBSS was added to the cellsso that the density was 1E08 cells/500 μl or less, and the cells weresuspended. Positive fractions of cells to which MACS streptavidin beadswere bound were collected from the cell suspensions using the autoMACSPro Separator.

A solution of biotinylated HMGB1 diluted to 500 nM with HBSS wasprepared again and added to the collected cells so that the density was1E08 cells/100 μl or less, and the cells were suspended. After 30minutes of incubation on ice, the cells were washed with 50 ml of HBSS.Solutions of streptavidin-FITC (BD) and mouse anti-rabbit IgG-PE(Southern Biotech) diluted with HBSS were prepared, and added to thecells so that the density was 1E08 cells/100 or less, and the cells weresuspended. After incubating the cell suspensions on ice for 30 minutes,the cells were washed with 50 ml of HBSS. Next, HBSS was added to thecells in such a way that the density was 1E07 cells/100 μl or less, andthey were suspended. From the cell suspensions, cells were collectedusing FACSAria (BD) with a gate for the fraction whose FITC and PEfluorescence intensities were both high. Upon collection, MBSS (20 mMMES, 5.3 mM KCl, 0.4 mM KH₂PO₄, 4.2 mM NaHCO₃, 0.3 mM Na₂HPO₄, 0.1% BSA,2 mM EDTA, 5 mM glucose, 138 mM NaCl, pH 5.8) was added to tubes for thecollection. Then, this was allowed to stand in the MBSS solution for 30minutes. In the case of antibodies that readily dissociate from anantigen by decrease in pH or Ca²⁺ ion concentration, when left in MBSSat low pH and in the presence of EDTA, the antigen dissociates,resulting in decreased FITC fluorescence intensity. After being allowedto stand for 30 minutes, the cells were collected using FACSAria again,while setting a gate for cell populations whose PE fluorescenceintensity was the same as the first sorting but the FITC fluorescenceintensity was lower than the first sorting. The result is shown inFIG. 1. (A) shows a dot plot result for the first sorting. The cellswithin the gate indicated as 1 were collected. (B) shows a dot plotresult for the second sorting. In the second sorting, the cells werecollected separately from gates 1, 2, and 3.

When performing this cell collection method, it is unnecessary toincrease the scale of subsequent screening for antibodies whosedissociation ability is altered depending on the change in pH or Ca²⁺ion concentration. Thus, it is possible to efficiently isolateantibodies whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration.

Culture of B Cells

The collected cells were seeded onto a 96-well microtiter plate at onecell or less/well. Activated rabbit T cell conditioned medium was addedat a final concentration of 5%, and EL4 cells (European Collection ofCell Cultures) were added at about 25000 cells/well. The activatedrabbit T cell conditioned medium was prepared as follows: thymusescollected from rabbits were filtered through 70 μm Cell Strainers (BDFalcon) using the plunger of a 5-ml syringe; the cells thus preparedwere cultured in RPMI-1640 containing PHA (Roche), phorbol 12-myristate13-acetate (Sigma), and 2% FBS; and the culture supernatants were frozenand stored until use at −70° C. or below. The EL4 cells to be used asfeeder cells were cultured at 37° C. under 5% CO₂ for two hours or moreafter adding mitomycin C (Sigma) at 10 μg/ml to stop the cell growth.After leaving the culture for 5 to 7 days at 37° C. under 5% CO₂, aportion of the supernatants containing secreted antibodies wascollected. Using the collected supernatants, the antibodies wereassessed for their HMGB1 binding by the method described below. Thecells were allowed to stand at 37° C. under 5% CO₂ until assessment ofthe antibodies for their HMGB1 binding.

Screening of Culture Supernatants for Monoclonal Antibodies with DesiredSpecificity

Screening was carried out by the ELISA method for the antigenrecognition of an antibody. A streptavidin-coated 384-well plate wasprepared and biotinylated HMGB1 was captured. Culture supernatantscontaining secreted antibodies were added to the plate. After leavingone hour at room temperature, this was washed three times with 80 μl ofTBS (TAKARA) containing 2 mM CaCl₂ and 0.05% Tween-20, and then adilution solution prepared by 40000-fold diluting Goat anti-rabbit IgGFc HRP conjugate (BETHYL) with TBS containing 2 mM CaCl₂ was aliquotedthereto, and this was allowed to stand at room temperature for one hour.This was washed three times with 80 μl of TBS (pH 7.4) containing 2 mMCaCl₂ and 0.05% Tween-20, and a chromogenic substrate (ABTS peroxidasesubstrate (KPL)) was added thereto at 40 μl/well. After one hour ofincubation, the absorbance at 405 nm was determined with SpectraMax fromMolecular Device. The measurement result for the absorbance at 405 nmwas analyzed to determine the wells with secreted antibodies thatrecognize the HMGB1 protein.

Screening for Antibodies Whose Dissociation Ability is Altered Dependingon the Change in pH or Ca²⁺ Ion Concentration

ELISA was performed using culture supernatants to assess the presence ofpH/Ca-dependent dissociation. A goat anti-rabbit IgG-Fc (BETHYL) dilutedto 1 μg/ml with PBS(−) was added to a 384-well MAXISorp (Nunc). Theplate was allowed to stand at room temperature for one hour or more.Then, the goat anti-rabbit IgG-Fc diluted with PBS(−) was removed fromthe plate, and TBS (pH 7.4) containing 1% BSA and 2 mM CaCl₂ was addedthereto. The plate was allowed to stand for one hour or more. TBS (pH7.4) containing 1% BSA and 2 mM CaCl₂ was removed from the plate, andculture supernatants were added thereto. In this step, when assessingantibodies whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration, each type of B cell culture supernatantwas aliquoted to two wells in the ELISA plate. Culture supernatants wereadded, and the plate was allowed to stand at room temperature for onehour or more, or at 4° C. overnight, to allow the goat anti-rabbitIgG-Fc to trap antibodies in the culture supernatants. Then, the platewas washed three times with 80 μl of TBS (pH 7.4) containing 2 mM CaCl₂and 0.05% Tween-20, and biotinylated HMGB1 was added thereto. The platewas allowed to stand at room temperature for one hour or more. Thisallowed biotinylated HMGB1 to bind to rabbit antibodies trapped by thegoat anti-rabbit IgG-Fc. The plate was washed three times with 80 μl ofTBS (pH 7.4) containing 2 mM CaCl₂ and 0.05% Tween-20 to wash offbiotinylated HMGB1 that were not bound to rabbit antibodies. Then, 20 mMMES (pH 7.4) containing 150 mM NaCl and 2 mM CaCl₂ (Buffer A) was addedto one of the above two wells containing the same culture supernatant,and 20 mM MES (pH 5.8) containing 150 mM NaCl and 2 mM EDTA (Buffer B)was added to the other. The plate was allowed to stand at 37° C. orbelow for one hour or more. In the presence of Buffer A or Buffer B,biotinylated human HMGB1 dissociated from rabbit antibodies. When, dueto its property, an antibody readily dissociates from an antigen at lowpH, or Ca²⁺ ion is required to maintain the binding of the antibody tothe antigen, the antigen more readily dissociated from the antibody on awell containing Buffer B than a well containing Buffer A. The plate waswashed three times with 80 μl of TBS (pH 7.4) containing 2 mM CaCl₂ and0.05% Tween-20. Then, 25 ng/ml of streptavidin-HRP (Genscript) preparedwith TBS containing 2 mM CaCl₂ was added, and the plate was allowed tostand at room temperature for one hour. Streptavidin-HRP bound tobiotinylated HMGB1 that remained without dissociating from the antibody.The plate was washed three times with 80 μl of TBS (pH 7.4) containing 2mM CaCl₂ and 0.05% Tween-20, and a chromogenic substrate (ABTSperoxidase substrate) was added thereto. After one hour of incubation,the absorbance at 405 nm was measured with SpectraMax from MolecularDevice. Based on the analysis of the result of absorbance measurement at405 nm, when the intensity of color development in the well to whichBuffer A was added was greater than that in the well to which Buffer Bwas added, the antibody in the culture supernatant was thought to be anantibody whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration.

This ELISA system has two features. One is that, for each B cell culturesupernatant, two wells of the culture supernatant are prepared in anELISA plate; and the antibody is allowed to bind to the antigen; andthen, for dissociation of the antigen from the antibody, incubation iscarried out under a condition at about pH 7.0 or above and a Ca²⁺ ionconcentration of about 1 mM or higher in one well, and under a conditionat about pH 6.0 or below and a low Ca²⁺ ion concentration in the otherwell. The presence of this incubation step enables efficient screeningfor antibodies whose dissociation ability is altered depending on thechange in pH or Ca²⁺ ion concentration. The other feature is that rabbitantibodies in the culture supernatant are trapped by the goatanti-rabbit IgG-Fc, and antigens are allowed to bind thereto. Whenrabbit antibodies are reacted after binding antigens to a plate, anantibody strongly bind to the antigens immobilized onto the plate withthe two arms, and the binding reaction is strong. Thus, even antibodiesthat show pH/Ca-dependent dissociation are less likely to dissociate,and one can only obtain those which exhibit strong dependence. Incontrast, when rabbit antibodies in the culture supernatant are trappedby the goat anti-rabbit IgG-Fc and antigens are allowed to bind thereto,the antigen/antibody binding is likely to occur in a one-to-one fashion.In this case, the presence or absence of the pH/Ca dependence can bedetermined even when the change in the dissociation ability depending onthe change in pH or Ca²⁺ ion concentration is small.

The result is shown in FIGS. 2 and 3. FIG. 2 is a dot plot for theresult of screening for antibodies whose dissociation ability is altereddepending on the change in pH or Ca²⁺ ion concentration. The Y axisindicates OD (405 nm) values under conditions of incubation with 20 mMMES (pH 7.4) containing 150 mM NaCl and 2 mM CaCl₂. The X axis indicatesOD (405 nm) values under conditions of incubation with 20 mM MES (pH5.8) containing 150 mM NaCl and 2 mM EDTA. (A) shows the result ofscreening for antibodies whose dissociation ability is altered dependingon the change in pH or Ca²⁺ ion concentration by culturing B cells fromgate 1 shown in FIG. 1(B). (B) shows the result of screening forantibodies whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration by culturing B cells from gate 2 shownin FIG. 1(B). (C) shows the result of screening for antibodies whosedissociation ability is altered depending on the change in pH or Ca²⁺ion concentration by culturing B cells from gate 3 shown in FIG. 1(B).The antibody that readily dissociates from the antigen when incubated atpH 5.8 in the presence of 2 mM EDTA as compared to when incubated at pH7.4 in the presence of 2 mM Ca²⁺ is indicated by circle. The antibodyfor which the value is not significantly altered between conditionscomprising pH 5.8 and 2 mM EDTA and pH 7.4 and 2 mM Ca²⁺ is indicated bysquare. FIG. 3 is a graphic representation of the numbers of clonesshown in FIGS. 2(A), 2(B), and 2(C). The gray area indicates the numberof clones that did not exhibit changes in antibody dissociation fromantigens even when pH or the Ca²⁺ ion concentration is altered(indicated by square in (A), (B), and (C)). The dark area indicates thenumber of clones of antibodies that readily dissociate from antigenswhen pH or the Ca²⁺ ion concentration is decreased (indicated by circlein (A), (B), and (C)). When antigen-binding B cells are collected, ofthe antigen-binding antibodies, the proportion of antigen-bindingantibodies whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration is about several percent at most, asseen in the result of (A). On the other hand, when B cells expressingantibodies whose dissociation ability is altered depending on the changein pH or Ca²⁺ ion concentration are enriched and collected, theproportion of antigen-binding antibodies whose dissociation ability isaltered depending on the change in pH or Ca²⁺ ion concentration, of theantigen-binding antibodies, can be increased up to several tens percent,as seen in the result of (C). This collection method allows efficientisolation of very rare antibodies whose dissociation ability is altereddepending on the change in pH or Ca²⁺ ion concentration and which havephysiological activity.

Sequence Identification of Variable Region L and H Chains from B Cellsand Expression of Recombinant Antibodies

Based on the result of screening for monoclonal antibodies havingdesired specificity, or for antibodies whose dissociation ability isaltered depending on the change in pH or Ca²⁺ ion concentration, cellsand culture supernatants were collected from a cell culture plateincubated at 37° C. under 5% CO₂, and transferred to a fresh 96-wellplates by MS2000 (J-Tek). From the plate containing the collected cellsand culture supernatants, the supernatants alone were transferred toanother 96-well plate. Meanwhile, the plate containing the cellscollected by MS2000 was frozen and stored at −70° C. or below. Toconstruct antibody expression vectors, antibody cDNAs were prepared fromthe cells frozen at −70° C. PCR primers to isolate the cDNAs weredesigned so that they anneal to the conserved regions in the sequence ofrabbit immunoglobulin (the H region and the L region). Antibody cDNAswere obtained by two collection steps using nested PCR. RNA was purifiedusing the MagMax 96 RNA Purification Kit for microarray (Ambion). Usingthe purified RNA, reverse transcription and first PCR was carried outwith the OneStep RT-PCR Kit (TAKARA). The primer sequences used areshown in Table 11. Then, the product of first PCR was subjected tonested PCR with PrimeSTAR HS (TAKARA). The primer sequences used for PCRare shown in Table 11. In the table, R represents a nucleotide mixtureof A and G; V represents a nucleotide mixture of A, C, and G; Wrepresents a nucleotide mixture of A and T; and Y represents anucleotide mixture of C and T.

TABLE 11  first H CHAIN Forward AS215 AGRACCCAGCATGGACAYVA PCR(SEQ ID NO: 8) Reverse AS217 GGAYRGWATTTATTYGCCAC RCACA (SEQ ID NO: 9)L CHAIN Forward AS219 AGACRCTCACCATGGAGACT (SEQ ID NO: 10) Reverse AS221ACTGGCTCCGGGAGGTA (SEQ ID NO: 11) nested H CHAIN Forward AS365CACCATGGAGACTGGGC PCR (SEQ ID NO: 12) Reverse AS368 GGAGGAGACGGTGAC(SEQ ID NO: 13) L CHAIN Forward AS359 ATGGACACGAGGGCCC (SEQ ID NO: 14)Reverse AS362 TTTGACCACCACCTCGGTC (SEQ ID NO: 15)

Cassette vectors were constructed by inserting the sequences of antibodyconstant regions into an animal cell expression vector, and they wereused to construct antibody expression vectors. The following two typesof cassette vectors were constructed: a vector carrying the sequence ofthe H chain constant region of a rabbit antibody; and a vector carryingthe sequence of the L chain constant region of a rabbit antibody. Thevector carrying the sequence of an H chain constant region has aninserted ampicillin resistance gene, while the vector carrying thesequence of an L chain constant region has an inserted kanamycinresistance gene. These two types of vectors have a partial sequenceoverlap with the nested PCR primer sequences. Using the In-Fusion PCRcloning Kit (Clontech), the nested PCR products are incorporated intothe cassette vectors introduced with the sequences of the antibodyconstant regions to construct expression vectors containing thefull-length rabbit antibody genes. The nested PCR products were insertedinto vectors using an In-Fusion PCR cloning Kit from Clontech, and thentransformed into bacteria for plasmid transmission and production. Thetransformed bacteria were cultured in LB media containing ampicillin orkanamycin. After purification of plasmids from the grown bacteria usingthe 96-well EndoFree ezFilter Plasmid Miniprep Kit (Biomiga), antibodieswere prepared according to Reference Example 1.

Example 2 Determination of the Affinity and pH/Ca Dependency ofAnti-HMGB1 Antibody Preparation of MedG4-IgG1 Antibody

MedG4H-IgG1 (SEQ ID NO: 36) and MedG4L-CK (SEQ ID NO: 37) were designedby linking the VH region (WO2007/084253, SEQ ID NO: 19) and VL region(WO2007/084253, SEQ ID NO: 17) of the G4 antibody described inWO2007/084253 to human IgG1 constant region and human Igκ constantregion, respectively. To prepare MedG4-IgG1, which is an anti-HMGB1antibody, DNAs encoding those described above were constructed usinggenetic engineering techniques, and expressed in animal cells by amethod known to those skilled in the art.

Assessment of Prepared Antibodies for the pH- and pH/Ca-DependentBinding Ability to Human HMGB1

Prepared antibodies were assessed for the presence of pH- andpH/Ca-dependent binding ability using BiacoreT100 and T200 (GEHealthcare). The plasma condition was set to be pH 7.4 and a calcium ionconcentration of 1.2 mM. Two types of intraendosomal conditions were setto be: pH 5.8 and a calcium ion concentration of 1.2 mM; and pH 5.8 anda calcium ion concentration of 3 μM. An appropriate amount of Protein A(Invitrogen) was immobilized onto Sensor chip CM4 (GE Healthcare) by theamine coupling method and antibodies of interest were captured thereon.The antigen used was human HMGB1. Measurements were carried out usingthree types of running buffers (#1: 20 mmol/l ACES, 150 mmol/l NaCl,0.05% (w/v) Tween20, 2 mmol/l CaCl₂, pH 7.4; #2: 20 mmol/l ACES, 150mmol/l NaCl, 0.05% (w/v) Tween20, 2 mmol/l CaCl₂, pH 5.8; #3: 20 mmol/lACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, 3 μmol/l CaCl₂, pH 5.8).Human HMGB1 was diluted using the respective running buffers.

HMG233-IgG1, HMG236-IgG1, HMG481-IgG1, and HMG487-IgG1

Antibodies diluted with a running buffer were captured onto a sensorchip by injecting at a flow rate of 10 μl/min for one minute. Then, asolution of diluted human HMGB1 (500 nM) and a running buffer (as areference solution) were injected at a flow rate of 10 μl/min for oneminute to interact with the captured antibodies. Then, a running bufferwas injected at a flow rate of 10 μl/min for one minute to observe thedissociation of human HMGB1. Finally, 10 mmol/l glycine-HCl (pH 1.5) wasinjected at a flow rate of 30 μl/min for 30 seconds to regenerate thesensor chip.

Sensorgrams obtained by the measurement are shown in FIGS. 4-1 and 4-2,in which the quantity of each antibody captured has been converted to100 RU. Since they are a box-shaped sensorgram with rapid convergence toan equilibrium state, the equilibrium value (i.e., binding amount)during injection of human HMGB1 reflects the dissociation constant KD(M). As to HMG233-IgG1 and HMG236-IgG1, each antibody showed asignificant decrease in the amount of binding to human HMGB1 under thecondition of pH 5.8 and 3 μM Ca as compared to the conditions of pH 7.4and 1.2 mM Ca and of pH 5.8 and 1.2 mM Ca. Regarding HMG481-IgG1 andHMG487-IgG1, each antibody exhibited a significant decrease in theamount of binding to human HMGB1 under the conditions of pH 5.8 and 1.2mM Ca, and of pH 5.8 and 3 μM Ca as compared to the condition of pH 7.4and 1.2 mM Ca.

HMG446-IgG1 and MedG4-IgG1

With respect to HMG446-IgG1 and MedG4-IgG1, an antibody diluted with arunning buffer was captured onto a sensor chip by injecting at a flowrate of 10 μl/min for one minute, and then a solution of diluted humanHMGB1 and a running buffer (as a reference solution) were injected at aflow rate of 10 μl/min for one minute to interact with the capturedantibody. Next, a running buffer was injected at a flow rate of 10μl/min for two minutes to observe the dissociation of human HMGB1.Finally, 10 mmol/l glycine-HCl (pH 1.5) was injected at a flow rate of30 μl/min for 30 seconds to regenerate the sensor chip.

Regarding MedG4-IgG1, a sensorgram obtained by the measurement wasanalyzed by curve fitting. A 1:1 binding model is used for the reactionmodel equation. The binding rate constant ka (1/Ms) and dissociationrate constant kd (1/s), which are kinetic parameters, were calculated,and based on the values, the dissociation constant KD (M) was calculatedfor each antibody for dissociation from human HMGB1. RegardingHMG446-IgG1, the dissociation constant KD (M) was calculated by applyinga steady state affinity model to the sensorgram obtained by themeasurement. Each parameter was calculated using Biacore T200 EvaluationSoftware (GE Healthcare). Meanwhile, the pH dependence was determined bydividing KD (M) at pH 5.8 and 1.2 mM Ca by KD (M) at pH 7.4 and 1.2 mMCa, whereas the pH/Ca dependence was determined by dividing KD (M) at pH5.8 and 3 μM Ca by KD (M) at pH 7.4 and 1.2 mM.

The analysis result is summarized in Table 12. The KD (M) of HMG446-IgG1at pH 7.4 and 1.2 mM Ca was calculated to be 220 nM. The KD (M) to humanHMGB1 was increased by 50 times (the affinity was reduced by 50 times)by the change from pH 7.4, 1.2 mM Ca to pH 5.8, 1.2 mM Ca, and it wasincreased by 82 times (the affinity was reduced by 82 times) by thechange to pH 5.8, 3 μM Ca. This demonstrates that the affinity for humanHMGB1 is reduced under the intraendosomal condition compared to theplasma condition. Meanwhile, the KD (M) of MedG4-IgG1 was calculated tobe 96 nM at pH 7.4 and 1.2 mM Ca, and 15 nM at pH 5.8 and 1.2 mM Ca and3 μM Ca. This demonstrates that the affinity for human HMGB1 isincreased under the intraendosomal condition compared to the plasmacondition, suggesting that MedG4-IgG1 is less likely to release HMGB1 inthe endosome.

TABLE 12 ANTIBODY NAME CONDITION K_(D) (M) DEPENDENCY MedG4 pH7.4, 1.2mM Ca 96 nM pH 5.8, 1.2 mM Ca 15 nM 0.15 TIMES (pH-DEPENDENT) pH 5.8, 3μM Ca 15 nM 0.15 TIMES (pH/Ca-DEPENDENT) HMG446 pH 7.4, 1.2 mM Ca 220nM  pH 5.8, 1.2 mM Ca 1.1 μM    50 TIMES (pH-DEPENDENT) pH 5.8, 3 μM Ca1.8 μM    82 TIMES (pH/Ca-DEPENDENT)

Example 3 Assessment for the Binding Between HMGB1 and the Cell SurfaceReceptor ELISA for the Binding of HMGB1 to RAGE-Fc

A solution containing 5 μg/ml recombinant human RAGE-Fc fusion protein(R&D SYSTEMS) in PBS was added to each well of an ELISA plate at 20μl/well. The plate was incubated at 4° C. overnight. Then, the plate wasblocked with 100 μl of 5% skim milk at 37° C. for one hour, and washedfour times with PBS/Tween. In another plate, 4 μg/ml HMGB1 waspre-incubated with 100 μg/ml anti-HMGB1 antibody or a buffer in thepresence of 2.5% skim milk at room temperature for one hour, and thiswas transferred to the RAGE-coated, blocked plate. Then, the plate wasincubated at 4° C. overnight, and washed four times with PBS/Tween. Todetect HMGB1 bound to the immobilized RAGE-Fc, a peroxidase-labeledmouse anti-HMGB1 monoclonal antibody was added to each well, and theplate was incubated at room temperature for two hours. Then, the platewas washed five times, and 20 μl of the chromogenic agent TMB was addedthereto. The absorbance at 450 nm in the plate was measured.

It was confirmed that the anti-HMGB1 antibody does not inhibit thebinding between HMGB1 and the peroxidase-labeled mouse anti-HMGB1monoclonal antibody used for detection, by allowing the anti-HMGB1antibody to compete the peroxidase-labeled mouse anti-HMGB1 monoclonalantibody in an HMGB1-immobilized plate.

ELISA for the Binding of HMGB1 to TLR4/MD-2

A solution of 5 μg/ml recombinant human TLR4/MD-2 protein (R&D SYSTEMS)in PBS was added to each well of an ELISA plate at 20 μl/well. The platewas incubated at 4° C. overnight. Then, the plate was blocked with 100μl of 5% skim milk at 37° C. for one hour, and washed four times withPBS/Tween. In another plate, 10 μg/ml HMGB1 was pre-incubated with 100μg/ml anti-HMGB1 antibody or a buffer in the presence of 2.5% skim milkat room temperature for one hour, and this was transferred to theTLR4/MD-2-coated, blocked plate. Then, the plate was incubated at roomtemperature for two hours, and washed four times with PBS/Tween. Todetect HMGB1 bound to the immobilized TLR4/MD-2, 1 μg/mlperoxidase-labeled mouse anti-HMGB1 monoclonal antibody was added toeach well, and the plate was incubated at room temperature for twohours. Then, the plate was washed five times with PBS/Tween, and 20 μlof the chromogenic agent TMB was added thereto. The absorbance at 450 nmin the plate was measured.

It was confirmed that the anti-HMGB1 antibody does not inhibit thebinding between HMGB1 and the peroxidase-labeled mouse anti-HMGB1monoclonal antibody used for detection, by allowing the anti-HMGB1antibody to compete the peroxidase-labeled mouse anti-HMGB1 monoclonalantibody in an HMGB1-immobilized plate.

Results

Both RAGE and TLR4 have been identified as putative receptors for HMGB1.Some anti-HMGB1 antibodies were assessed by ELISA assay for theirability to inhibit the binding between HMGB1 and the RAGE-Fc fusionproduct or TLR4/MD-2 fusion product.

By setting the value determined under the condition in the absence ofthe anti-HMGB1 antibody as 100, a relative value was determined for eachof RAGE and TLR4 from the value determined under each condition in thepresence of the anti-HMGB1 antibody. The values are shown in FIGS. 5 and6. Anti-HMGB1 antibodies with a value less than 100 were determined tohave the ability to inhibit the binding to each receptor. Of theantibodies subjected to RAGE ELISA, HMG233-IgG1 and HMG236-IgG1inhibited the binding of HMGB1 to RAGE. The percent inhibition was 53.1%for HMG233-IgG1, and 64.1% for HMG236-IgG1. Of the antibodies subjectedto TLR4/MD-2 ELISA, HMG481-IgG1, HMG487-IgG1, and HMG446-IgG1 inhibitedthe binding of HMGB1 to TLR4/MD-2. The percent inhibition was 93.5% forHMG481-IgG1, 75.7% for HMG487-IgG1, and 81.3% for HMG446-IgG1.HMG233-IgG1 and HMG236-IgG1 did not inhibit the binding between HMGB1and TLR4/MD-2. Meanwhile, HMG481-IgG1, HMG487-IgG1, and HMG446-IgG1 didnot inhibit the binding between HMGB1 and RAGE.

The present invention demonstrated that, of anti-HMGB1 antibodies, someantibodies inhibited the binding of HMGB1 to RAGE but not the binding toTLR4, and that some antibody inhibited the binding of HMGB1 to TLR4/MD-2but not the binding to RAGE.

Example 4 Improvement of the Effect to Accelerate the Elimination ofHuman HMGB1 by the pH-Dependent Anti-Human HMGB1 Antibody In Vivo TestUsing Normal Mice

Human HMGB1 and anti-human HMGB1 antibody were simultaneouslyadministered to normal mice (C57BL/6J mouse, Charles River Japan) toassess the in vivo kinetics of human HMGB1 and the anti-human HMGB1antibody. A mixed solution of human HMGB1 (0.1 mg/ml) and the anti-humanHMGB1 antibody (1 mg/ml MedG4-IgG1, 2.05 mg/ml HMG446) was administeredat 10 ml/kg once into the tail vein. The antibody concentration in thismixed solution was adopted as a concentration that allows 99.0% or moreof human HMGB1 contained in the solution to bind to the antibody. Bloodwas collected at 5 minutes, 10 minutes, 15 minutes, one hour, 4 hours, 2days, and 7 days after administration. The collected blood was allowedto stand for two hours, and then centrifuged at 12,000 rpm and 4° C. for5 minutes to obtain sera. The isolated sera were stored in a freezer setat −20° C. or below until use. Herein, MedG4-IgG1 is sometimes referredto as “med G4”, while HMG446-IgG1 is sometimes referred to as“HMG446-G1”.

Determination of Anti-Human HMGB1 Antibody Concentration in Serum byELISA

Anti-human HMGB1 antibody concentrations in mouse sera were determinedby ELISA. First, to prepare an anti-human IgG-immobilized plate,Anti-Human IgG (γ-chain specific) F(ab′)2 Fragment of Antibody (SIGMA)was aliquoted into a Nunc-Immuno Plate, MaxiSorp (Nalge nuncInternational), and the plate was allowed to stand at 4° C. overnight.Standard curve samples (serum concentrations of 3.2, 1.6, 0.8, 0.4, 0.2,0.1, and 0.05 μg/ml) and assay samples of mouse serum diluted 100 timesor more were prepared. 150 μl of 2000 ng/ml human HMGB1 was added to 150μl of the standard curve samples and assay samples. This was allowed tostand at room temperature for one hour, and then aliquoted into theanti-human IgG-immobilized plate. The plate was allowed to stand at roomtemperature for one hour. Then, Goat Anti-Human IgG (γ chain specific)Biotin (BIOT) Conjugate (Southern Biotech Association) was reacted atroom temperature for one hour. Next, Streptavidin-PolyHRP80(Stereospecific Detection Technologies) was reacted at room temperaturefor one hour. Chromogenic reaction was performed using TMB One ComponentHRP Microwell Substrate (BioFX Laboratories) as a substrate. Afterterminating the reaction with 1N sulfuric acid (Showa Chemical), theabsorbance at 450 nm was measured with a microplate reader. Theanti-human HMGB1 antibody concentrations in mouse sera were determinedbased on the absorbance of the standard curve using the analysissoftware SOFTmax PRO (Molecular Devices). A time course of anti-humanHMGB1 antibody concentration in the sera of mice after intravenousadministration, which was measured by the above method, is shown in FIG.8.

Measurement of Human HMGB1 Concentration in Serum by ELISA

Human HMGB1 concentrations in mouse sera were measured using HMGB1 ELISAKit II (shino-test). Standard curve samples with a serum concentrationof 12800, 6400, 3200, 1600, 800, 400, or 200 μg/ml, and assay samples ofmouse sera diluted 100 times or more were prepared, and mixed with anequal volume of 40 μg/ml HMG446 solution (in the presence of HMG446-IgG1or HMG446-F1) or 20 μg/ml MedG4-IgG1 solution (in the presence ofMedG4-IgG1). After one hour of incubation at room temperature, themixtures were aliquoted in the attached, immobilized plate. The platewas incubated at 37° C. for 20 to 24 hours. Then, the attached, labeledantibody solution was reacted at 25° C. for two hours. After 30 minutesof reaction with a chromogenic reagent, the reaction was stopped byadding a stop solution. Then, the absorbance at 450 nm was measuredusing a microplate reader. The human HMGB1 concentrations in mouse serawere calculated based on the absorbance of the standard curve using theanalysis software SOFTmax PRO (Molecular Devices). A time course ofhuman HMGB1 concentration in the sera of mice after intravenousadministration, which was measured by the above method, is shown in FIG.7.

The Effect of pH/Ca-Dependent Binding to Human HMGB1

HMG446-IgG1 whose human HMGB1-binding activity is decreased at acidic pHor low calcium ion concentration and MedG4-IgG1 whose humanHMGB1-binding activity is not decreased at acidic pH or low calcium ionconcentration were tested in vivo, and the results were compared to eachother. As shown in FIG. 8, the pharmacokinetics of the two antibodiesshowed linearity. Meanwhile, as shown in FIG. 7, it was revealed that,when HMGB1 was administered in combination with HMG446-IgG1 that bindsto human HMGB1 in a pH-dependent manner, HMG446-IgG1 accelerated theelimination of HMGB1 as compared to when MedG4-IgG1 was administered incombination with HMGB1. Thus, it was demonstrated that, by conferringthe pH-dependent human HMGB1-binding ability, the serum HMGB1concentration can be decreased by about 4.0 times one day afteradministration.

The Effect of FcRn Binding at Neutral Conditions (pH 7.4)

In addition to HMG446-IgG1, HMG446-F1 resulting from introducing anamino acid substitution into the IgG Fc region of HMG446-IgG1, wastested in vivo using mice. The test result was compared to that forHMG446-IgG1. As shown in FIG. 8, the serum antibody concentration ofHMG446-F1 whose mouse FcRn binding was enhanced under a neutralcondition (pH 7.4) was lower by about 1.2 times than that of HMG446-IgG115 minutes after administration.

As shown in FIG. 7, it was demonstrated that HMGB1, when administered incombination with HMG446-F1 whose mouse FcRn binding was enhanced under aneutral condition (pH 7.4), was eliminated more rapidly as compared towhen HMGB1 was administered in combination with HMG446-IgG1. HMG446-F1reduced the serum HMGB1 concentration by about 2.4 times in 15 minutesas compared to HMG446-IgG1. Thus, it was demonstrated that the serumhuman HMGB1 concentration can be reduced by conferring the mouseFcRn-binding ability under a neutral condition (pH 7.4). As describedabove, the antibody concentration in serum was reduced by conferring theability to bind to mouse FcRn under a neutral condition (pH 7.4).However, the achieved effect to decrease the serum HMGB1 concentrationexceeded the decrease in antibody concentration.

The above results suggest that, by administering an antibody whosebinding activity to human HMGB1 is reduced under an acidic pH or lowcalcium ion concentration condition, the elimination of human HMGB1 canbe accelerated as compared to when administering an antibody whosebinding activity to human HMGB1 is not reduced at an acidic pH or lowcalcium ion concentration condition, and this effect is enhanceddepending on the mouse FcRn-binding ability under a neutral condition(pH 7.4).

Example 5 Production of Various Antibody Fc Variants with IncreasedBinding Affinity for Human FcRn at Neutral pH Production of Fc Variants

To increase the binding affinity for human FcRn in a neutral pH range,various substitutions were introduced into the antibody Fv4-IgG1 thatcomprise the H chain and L chain which are, respectively, VH3-IgG1 andVL3-CK described as SEQ ID NOs: 6 and 7 in WO2009/125825. Specifically,the amino acid substitutions shown in Table 13-1 to 13-14 wereintroduced into the heavy chain constant region of Fv4-IgG1 to create Fcvariants (the amino acid numbers for mutation sites are shown accordingto EU numbering). Amino acid substitutions were introduced according tothe method known to those skilled in the art, which is described inReference Example 1.

Variants containing the prepared heavy chains and the light chain L(WT)described as SEQ ID NO: 5 in WO2009/125825 were expressed and purifiedby the method known to those skilled in the art, which is described inReference Example 1.

Assessment of Human FcRn Binding

The binding between the antibody and human FcRn under a neutralcondition (pH 7.0) was analyzed with Biacore. The results are shown inTable 13-1 to 13-14.

TABLE 13-1 VARIANT NAME KD (M) AMINO ACID SUBSTITUTION F1  8.10E−07N434W F2  3.20E−06 M252Y/S254T/T256E F3  2.50E−06 N434Y F4  5.80E−06N434S F5  6.80E−06 N434A F7  5.60E−06 M252Y F8  4.20E−06 M252W F9 1.40E−07 M252Y/S254T/T256E/N434Y F10 6.90E−08 M252Y/S254T/T256E/N434WF11 3.10E−07 M252Y/N434Y F12 1.70E−07 M252Y/N434W F13 3.20E−07M252W/N434Y F14 1.80E−07 M252W/N434W F19 4.60E−07 P257L/N434Y F204.60E−07 V308F/N434Y F21 3.00E−08 M252Y/V308P/N434Y F22 2.00E−06M428L/N434S F25 9.20E−09 M252Y/S254T/T256E/V308P/N434W F26 1.00E−06I332V F27 7.40E−06 G237M F29 1.40E−06 I332V/N434Y F31 2.80E−06G237M/V308F F32 8.00E−07 S254T/N434W F33 2.30E−06 S254T/N434Y F342.80E−07 T256E/N434W F35 8.40E−07 T256E/N434Y F36 3.60E−07S254T/T256E/N434W F37 1.10E−06 S254T/T256E/N434Y F38 1.00E−07M252Y/S254T/N434W F39 3.00E−07 M252Y/S254T/N434Y F40 8.20E−08M252Y/T256E/N434W F41 1.50E−07 M252Y/T256E/N434Y F42 1.00E−06M252Y/S254T/T256E/N434A F43 1.70E−06 M252Y/N434A F44 1.10E−06M252W/N434A F47 2.40E−07 M252Y/T256Q/N434W F48 3.20E−07M252Y/T256Q/N434Y F49 5.10E−07 M252F/T256D/N434W F50 1.20E−06M252F/T256D/N434Y F51 8.10E−06 N434F/Y436H F52 3.10E−06H433K/N434F/Y436H F53 1.00E−06 I332V/N434W F54 8.40E−08 V308P/N434W F569.40E−07 I332V/M428L/N434Y F57 1.10E−05 G385D/Q386P/N389S F58 7.70E−07G385D/Q386P/N389S/N434W F59 2.40E−06 G385D/Q386P/N389S/N434Y F601.10E−05 G385H F61 9.70E−07 G385H/N434W F62 1.90E−06 G385H/N434Y F632.50E−06 N434F F64 5.30E−06 N434H F65 2.90E−07 M252Y/S254T/T256E/N434FF66 4.30E−07 M252Y/S254T/T256E/N434H F67 6.30E−07 M252Y/N434F F689.30E−07 M252Y/N434H F69 5.10E−07 M428L/N434W F70 1.50E−06 M428L/N434YF71 8.30E−08 M252Y/S254T/T256E/M428L/N434W F72 2.00E−07M252Y/S254T/T256E/M428L/N434Y

Table 13-2 is the continuation of Table 13-1.

TABLE 13-2 F73  1.70E−07 M252Y/M428L/N434W F74  4.60E−07M252Y/M428L/N434Y F75  1.40E−06 M252Y/M428L/N434A F76  1.00E−06M252Y/S254T/T256E/M428L/N434A F77  9.90E−07 T256E/M428L/N434Y F78 7.80E−07 S254T/M428L/N434W F79  5.90E−06 S254T/T256E/N434A F80  2.70E−06M252Y/T256Q/N434A F81  1.60E−06 M252Y/T256E/N434A F82  1.10E−06T256Q/N434W F83  2.60E−06 T256Q/N434Y F84  2.80E−07 M252W/T256Q/N434WF85  5.50E−07 M252W/T256Q/N434Y F86  1.50E−06 S254T/T256Q/N434W F87 4.30E−06 S254T/T256Q/N434Y F88  1.90E−07 M252Y/S254T/T256Q/N434W F89 3.60E−07 M252Y/S254T/T256Q/N434Y F90  1.90E−08 M252Y/T256E/V308P/N434WF91  4.80E−08 M252Y/V308P/M428L/N434Y F92  1.10E−08M252Y/S254T/T256E/V308P/M428L/N434W F93  7.40E−07 M252W/M428L/N434W F94 3.70E−07 P257L/M428L/N434Y F95  2.60E−07 M252Y/S254T/T256E/M428L/N434FF99  6.20E−07 M252Y/T256E/N434H F101 1.10E−07 M252W/T256Q/P257L/N434YF103 4.40E−08 P238A/M252Y/V308P/N434Y F104 3.70E−08M252Y/D265A/V308P/N434Y F105 7.50E−08 M252Y/T307A/V308P/N434Y F1063.70E−08 M252Y/V303A/V308P/N434Y F107 3.40E−08 M252Y/V308P/D376A/N434YF108 4.10E−08 M252Y/V305A/V308P/N434Y F109 3.20E−08M252Y/V308P/Q311A/N434Y F111 3.20E−08 M252Y/V308P/K317A/N434Y F1126.40E−08 M252Y/V308P/E380A/N434Y F113 3.20E−08 M252Y/V308P/E382A/N434YF114 3.80E−08 M252Y/V308P/S424A/N434Y F115 6.60E−06 T307A/N434A F1168.70E−06 E380A/N434A F118 1.40E−05 M428L F119 5.40E−06 T250Q/M428L F1206.30E−08 P257L/V308P/M428L/N434Y F121 1.50E−08M252Y/T256E/V308P/M428L/N434W F122 1.20E−07 M252Y/T256E/M428L/N434W F1233.00E−08 M252Y/T256E/V308P/N434Y F124 2.90E−07 M252Y/T256E/M428L/N434YF125 2.40E−08 M252Y/S254T/T256E/V308P/M428L/N434Y F128 1.70E−07P257L/M428L/N434W F129 2.20E−07 P257A/M428L/N434Y F131 3.00E−06P257G/M428L/N434Y F132 2.10E−07 P257I/M428L/N434Y F133 4.10E−07P257M/M428L/N434Y F134 2.70E−07 P257N/M428L/N434Y F135 7.50E−07P257S/M428L/N434Y F136 3.80E−07 P257T/M428L/N434Y F137 4.60E−07P257V/M428L/N434Y F139 1.50E−08 M252W/V308P/N434W F140 3.60E−08S239K/M252Y/V308P/N434Y F141 3.50E−08 M252Y/S298G/V308P/N434Y F1423.70E−08 M252Y/D270F/V308P/N434Y F143 2.00E−07 M252Y/V308A/N434Y F1455.30E−08 M252Y/V308F/N434Y

Table 13-3 is the continuation of Table 13-2.

TABLE 13-3 F147 2.40E−07 M252Y/V308I/N434Y F149 1.90E−07M252Y/V308L/N434Y F150 2.00E−07 M252Y/V308M/N434Y F152 2.70E−07M252Y/V308Q/N434Y F154 1.80E−07 M252Y/V308T/N434Y F157 1.50E−07P257A/V308P/M428L/N434Y F158 5.90E−08 P257T/V308P/M428L/N434Y F1594.40E−08 P257V/V308P/M428L/N434Y F160 8.50E−07 M252W/M428I/N434Y F1621.60E−07 M252W/M428Y/N434Y F163 4.20E−07 M252W/M428F/N434Y F164 3.70E−07P238A/M252W/N434Y F165 2.90E−07 M252W/D265A/N434Y F166 1.50E−07M252W/T307Q/N434Y F167 2.90E−07 M252W/V303A/N434Y F168 3.20E−07M252W/D376A/N434Y F169 2.90E−07 M252W/V305A/N434Y F170 1.70E−07M252W/Q311A/N434Y F171 1.90E−07 M252W/D312A/N434Y F172 2.20E−07M252W/K317A/N434Y F173 7.70E−07 M252W/E380A/N434Y F174 3.40E−07M252W/E382A/N434Y F175 2.70E−07 M252W/S424A/N434Y F176 2.90E−07S239K/M252W/N434Y F177 2.80E−07 M252W/S298G/N434Y F178 2.70E−07M252W/D270F/N434Y F179 3.10E−07 M252W/N325G/N434Y F182 6.60E−08P257A/M428L/N434W F183 2.20E−07 P257T/M428L/N434W F184 2.70E−07P257V/M428L/N434W F185 2.60E−07 M252W/I332V/N434Y F188 3.00E−06P257I/Q311I F189 1.90E−07 M252Y/T307A/N434Y F190 1.10E−07M252Y/T307Q/N434Y F191 1.60E−07 P257L/T307A/M428L/N434Y F192 1.10E−07P257A/T307A/M428L/N434Y F193 8.50E−08 P257T/T307A/M428L/N434Y F1941.20E−07 P257V/T307A/M428L/N434Y F195 5.60E−08 P257L/T307Q/M428L/N434YF196 3.50E−08 P257A/T307Q/M428L/N434Y F197 3.30E−08P257T/T307Q/M428L/N434Y F198 4.80E−08 P257V/T307Q/M428L/N434Y F2012.10E−07 M252Y/T307D/N434Y F203 2.40E−07 M252Y/T307F/N434Y F204 2.10E−07M252Y/T307G/N434Y F205 2.00E−07 M252Y/T307H/N434Y F206 2.30E−07M252Y/T307I/N434Y F207 9.40E−07 M252Y/T307K/N434Y F208 3.90E−07M252Y/T307L/N434Y F209 1.30E−07 M252Y/T307M/N434Y F210 2.90E−07M252Y/T307N/N434Y F211 2.40E−07 M252Y/T307P/N434Y F212 6.80E−07M252Y/T307R/N434Y F213 2.30E−07 M252Y/T307S/N434Y F214 1.70E−07M252Y/T307V/N434Y F215 9.60E−08 M252Y/T307W/N434Y F216 2.30E−07M252Y/T307Y/N434Y F217 2.30E−07 M252Y/K334L/N434Y F218 2.60E−07M252Y/G385H/N434Y F219 2.50E−07 M252Y/T289H/N434Y F220 2.60E−07M252Y/Q311H/N434Y

Table 13-4 is the continuation of Table 13-3.

TABLE 13-4 F221 3.10E−07 M252Y/D312H/N434Y F222 3.40E−07M252Y/N315H/N434Y F223 2.70E−07 M252Y/K360H/N434Y F225 1.50E−06M252Y/L314R/N434Y F226 5.40E−07 M252Y/L314K/N434Y F227 1.20E−07M252Y/N286E/N434Y F228 2.30E−07 M252Y/L309E/N434Y F229 5.10E−07M252Y/R255E/N434Y F230 2.50E−07 M252Y/P387E/N434Y F236 8.90E−07K248I/M428L/N434Y F237 2.30E−07 M252Y/M428A/N434Y F238 7.40E−07M252Y/M428D/N434Y F240 7.20E−07 M252Y/M428F/N434Y F241 1.50E−06M252Y/M428G/N434Y F242 8.50E−07 M252Y/M428H/N434Y F243 1.80E−07M252Y/M428I/N434Y F244 1.30E−06 M252Y/M428K/N434Y F245 4.70E−07M252Y/M428N/N434Y F246 1.10E−06 M252Y/M428P/N434Y F247 4.40E−07M252Y/M428Q/N434Y F249 6.40E−07 M252Y/M428S/N434Y F250 2.90E−07M252Y/M428T/N434Y F251 1.90E−07 M252Y/M428V/N434Y F252 1.00E−06M252Y/M428W/N434Y F253 7.10E−07 M252Y/M428Y/N434Y F254 7.50E−08M252W/T307Q/M428Y/N434Y F255 1.10E−07 M252W/Q311A/M428Y/N434Y F2565.40E−08 M252W/T307Q/Q311A/M428Y/N434Y F257 5.00E−07M252Y/T307A/M428Y/N434Y F258 3.20E−07 M252Y/T307Q/M428Y/N434Y F2592.80E−07 M252Y/D270F/N434Y F260 1.30E−07 M252Y/T307A/Q311A/N434Y F2618.40E−08 M252Y/T307Q/Q311A/N434Y F262 1.90E−07 M252Y/T307A/Q311H/N434YF263 1.10E−07 M252Y/T307Q/Q311H/N434Y F264 2.80E−07 M252Y/E382A/N434YF265 6.80E−07 M252Y/E382A/M428Y/N434Y F266 4.70E−07M252Y/T307A/E382A/M428Y/N434Y F267 3.20E−07M252Y/T307Q/E382A/M428Y/N434Y F268 6.30E−07 P238A/M252Y/M428F/N434Y F2695.20E−07 M252Y/V305A/M428F/N434Y F270 6.60E−07 M252Y/N325G/M428F/N434YF271 6.90E−07 M252Y/D376A/M428F/N434Y F272 6.80E−07M252Y/E380A/M428F/N434Y F273 6.50E−07 M252Y/E382A/M428F/N434Y F2747.60E−07 M252Y/E380A/E382A/M428F/N434Y F275 4.20E−08S239K/M252Y/V308P/E382A/N434Y F276 4.10E−08M252Y/D270F/V308P/E382A/N434Y F277 1.30E−07S239K/M252Y/V308P/M428Y/N434Y F278 3.00E−08M252Y/T307Q/V308P/E382A/N434Y F279 6.10E−08M252Y/V308P/Q311H/E382A/N434Y F280 4.10E−08S239K/M252Y/D270F/V308P/N434Y F281 9.20E−08M252Y/V308P/E382A/M428F/N434Y F282 2.90E−08M252Y/V308P/E382A/M428L/N434Y F283 1.00E−07M252Y/V308P/E382A/M428Y/N434Y F284 1.00E−07 M252Y/V308P/M428Y/N434Y F2859.90E−08 M252Y/V308P/M428F/N434Y F286 1.20E−07S239K/M252Y/V308P/E382A/M428Y/N434Y F287 1.00E−07M252Y/V308P/E380A/E382A/M428F/N434Y F288 1.90E−07M252Y/T256E/E382A/N434Y F289 4.80E−07 M252Y/T256E/M428Y/N434Y

Table 13-5 is the continuation of Table 13-4.

TABLE 13-5 F290 4.60E−07 M252Y/T256E/E382A/M428Y/N434Y F292 2.30E−08S239K/M252Y/V308P/E382A/M428I/N434Y F293 5.30E−08M252Y/V308P/E380A/E382A/M428I/N434Y F294 1.10E−07S239K/M252Y/V308P/M428F/N434Y F295 6.80E−07S239K/M252Y/E380A/E382A/M428F/N434Y F296 4.90E−07M252Y/Q311A/M428Y/N434Y F297 5.10E−07 M252Y/D312A/M428Y/N434Y F2984.80E−07 M252Y/Q311A/D312A/M428Y/N434Y F299 9.40E−08S239K/M252Y/V308P/Q311A/M428Y/N434Y F300 8.30E−08S239K/M252Y/V308P/D312A/M428Y/N434Y F301 7.20E−08S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y F302 1.90E−07M252Y/T256E/T307P/N434Y F303 6.70E−07 M252Y/T307P/M428Y/N434Y F3041.60E−08 M252W/V308P/M428Y/N434Y F305 2.70E−08M252Y/T256E/V308P/E382A/N434Y F306 3.60E−08 M252W/V308P/E382A/N434Y F3073.60E−08 S239K/M252W/V308P/E382A/N434Y F308 1.90E−08S239K/M252W/V308P/E382A/M428Y/N434Y F310 9.40E−08S239K/M252W/V308P/E382A/M428I/N434Y F311 2.80E−08S239K/M252W/V308P/M428F/N434Y F312 4.50E−07S239K/M252W/E380A/E382A/M428F/N434Y F313 6.50E−07S239K/M252Y/T307P/M428Y/N434Y F314 3.20E−07M252Y/T256E/Q311A/D312A/M428Y/N434Y F315 6.80E−07S239K/M252Y/M428Y/N434Y F316 7.00E−07 S239K/M252Y/D270F/M428Y/N434Y F3171.10E−07 S239K/M252Y/D270F/V308P/M428Y/N434Y F318 1.80E−08S239K/M252Y/V308P/M428I/N434Y F320 2.00E−08S239K/M252Y/V308P/N325G/E382A/M428I/N434Y F321 3.20E−08S239K/M252Y/D270F/V308P/N325G/N434Y F322 9.20E−08S239K/M252Y/D270F/T307P/V308P/N434Y F323 2.70E−08S239K/M252Y/T256E/D270F/V308P/N434Y F324 2.80E−08S239K/M252Y/D270F/T307Q/V308P/N434Y F325 2.10E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y F326 7.50E−08S239K/M252Y/D270F/T307Q/Q311A/N434Y F327 6.50E−08S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y F328 1.90E−08S239K/M252Y/D270F/V308P/M428I/N434Y F329 1.20E−08S239K/M252Y/D270F/N286E/V308P/N434Y F330 3.60E−08S239K/M252Y/D270F/V308P/L309E/N434Y F331 3.00E−08S239K/M252Y/D270F/V308P/P387E/N434Y F333 7.40E−08S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y F334 1.90E−08S239K/M252Y/D270F/V308P/N325G/M428I/N434Y F335 1.50E−08S239K/M252Y/T256E/D270F/V308P/M428I/N434Y F336 1.40E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/ N434Y F337 5.60E−08S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y F338 7.70E−09S239K/M252Y/D270F/N286E/V308P/M428I/N434Y F339 1.90E−08S239K/M252Y/D270F/V308P/L309E/M428I/N434Y F343 3.20E−08S239K/M252Y/D270F/V308P/M428L/N434Y F344 3.00E−08S239K/M252Y/V308P/M428L/N434Y F349 1.50E−07S239K/M252Y/V308P/L309P/M428L/N434Y F350 1.70E−07S239K/M252Y/V308P/L309R/M428L/N434Y F352 6.00E−07S239K/M252Y/L309P/M428L/N434Y F353 1.10E−06S239K/M252Y/L309R/M428L/N434Y F354 2.80E−08S239K/M252Y/T307Q/V308P/M428L/N434Y F356 3.40E−08S239K/M252Y/D270F/V308P/L309E/P387E/N434Y F357 1.60E−08S239K/M252Y/T256E/D270F/V308P/N325G/M428I/ N434Y F358 1.00E−07S239K/M252Y/T307Q/N434Y F359 4.20E−07 P257V/T307Q/M428I/N434Y F3601.30E−06 P257V/T307Q/M428V/N434Y F362 5.40E−08P257V/T307Q/N325G/M428L/N434Y F363 4.10E−08P257V/T307Q/Q311A/M428L/N434Y F364 3.50E−08P257V/T307Q/Q311A/N325G/M428L/N434Y

Table 13-6 is the continuation of Table 13-5.

TABLE 13-6 F365 5.10E−08 P257V/V305A/T307Q/M428L/N434Y F367 1.50E−08S239K/M252Y/E258H/D270F/T307Q/V308P/ Q311A/N434Y F368 2.00E−08S239K/M252Y/D270F/V308P/N325G/E382A/ M428I/N434Y F369 7.50E−08M252Y/P257V/T307Q/M428I/N434Y F372 1.30E−08S239K/M252W/V308P/M428Y/N434Y F373 1.10E−08S239K/M252W/V308P/Q311A/M428Y/N434Y F374 1.20E−08S239K/M252W/T256E/V308P/M428Y/N434Y F375 5.50E−09S239K/M252W/N286E/V308P/M428Y/N434Y F376 9.60E−09S239K/M252Y/T256E/D270F/N286E/V308P/ N434Y F377 1.30E−07S239K/M252W/T307P/M428Y/N434Y F379 9.00E−09S239K/M252W/T256E/V308P/Q311A/M428Y/ N434Y F380 5.60E−09S239K/M252W/T256E/N286E/V308P/M428Y/ N434Y F381 1.10E−07P257V/T307A/Q311A/M428L/N434Y F382 8.70E−08P257V/V305A/T307A/M428L/N434Y F386 3.20E−08 M252Y/V308P/L309E/N434Y F3871.50E−07 M252Y/V308P/L309D/N434Y F388 7.00E−08 M252Y/V308P/L309A/N434YF389 1.70E−08 M252W/V308P/L309E/M428Y/N434Y F390 6.80E−08M252W/V308P/L309D/M428Y/N434Y F391 3.60E−08M252W/V308P/L309A/M428Y/N434Y F392 6.90E−09S239K/M252Y/N286E/V308P/M428I/N434Y F393 1.20E−08S239K/M252Y/N286E/V308P/N434Y F394 5.30E−08S239K/M252Y/T307Q/Q311A/M428I/N434Y F395 2.40E−08S239K/M252Y/T256E/V308P/N434Y F396 2.00E−08S239K/M252Y/D270F/N286E/T307Q/Q311A/ M428I/N434Y F397 4.50E−08S239K/M252Y/D270F/T307Q/Q311A/P387E/ M428I/N434Y F398 4.40E−09S239K/M252Y/D270F/N286E/T307Q/V308P/ Q311A/M428I/N434Y F399 6.50E−09S239K/M252Y/D270F/N286E/T307Q/V308P/ M428I/N434Y F400 6.10E−09S239K/M252Y/D270F/N286E/V308P/Q311A/ M428I/N434Y F401 6.90E−09S239K/M252Y/D270F/N286E/V308P/P387E/ M428I/N434Y F402 2.30E−08P257V/T307Q/M428L/N434W F403 5.10E−08 P257V/T307A/M428L/N434W F4049.40E−08 P257A/T307Q/L309P/M428L/N434Y F405 1.70E−07P257V/T307Q/L309P/M428L/N434Y F406 1.50E−07P257A/T307Q/L309R/M428L/N434Y F407 1.60E−07P257V/T307Q/L309R/M428L/N434Y F408 2.50E−07 P257V/N286E/M428L/N434Y F4092.00E−07 P257V/P387E/M428L/N434Y F410 2.20E−07 P257V/T307H/M428L/N434YF411 1.30E−07 P257V/T307N/M428L/N434Y F412 8.80E−08P257V/T307G/M428L/N434Y F413 1.20E−07 P257V/T307P/M428L/N434Y F4141.10E−07 P257V/T307S/M428L/N434Y F415 5.60E−08P257V/N286E/T307A/M428L/N434Y F416 9.40E−08P257V/T307A/P387E/M428L/N434Y F418 6.20E−07S239K/M252Y/T307P/N325G/M428Y/N434Y F419 1.60E−07M252Y/T307A/Q311H/K360H/N434Y F420 1.50E−07M252Y/T307A/Q311H/P387E/N434Y F421 1.30E−07M252Y/T307A/Q311H/M428A/N434Y F422 1.80E−07M252Y/T307A/Q311H/E382A/N434Y F423 8.40E−08 M252Y/T307W/Q311H/N434Y F4249.40E−08 S239K/P257A/V308P/M428L/N434Y F425 8.00E−08P257A/V308P/L309E/M428L/N434Y F426 8.40E−08 P257V/T307Q/N434Y F4271.10E−07 M252Y/P257V/T307Q/M428V/N434Y F428 8.00E−08M252Y/P257V/T307Q/M428L/N434Y F429 3.70E−08 M252Y/P257V/T307Q/N434Y F4308.10E−08 M252Y/P257V/T307Q/M428Y/N434Y F431 6.50E−08M252Y/P257V/T307Q/M428F/N434Y F432 9.20E−07P257V/T307Q/Q311A/N325G/M428V/N434Y

Table 13-7 is the continuation of Table 13-6.

TABLE 13-7 F433 6.00E−08 P257V/T307Q/Q311A/N325G/N434Y F434 2.00E−08P257V/T307Q/Q311A/N325G/M428Y/N434Y F435 2.50E−08P257V/T307Q/Q311A/N325G/M428F/N434Y F436 2.50E−07P257A/T307Q/M428V/N434Y F437 5.70E−08 P257A/T307Q/N434Y F438 3.60E−08P257A/T307Q/M428Y/N434Y F439 4.00E−08 P257A/T307Q/M428F/N434Y F4401.50E−08 P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y F441 1.80E−07P257A/Q311A/M428L/N434Y F442 2.00E−07 P257A/Q311H/M428L/N434Y F4435.50E−08 P257A/T307Q/Q311A/M428L/N434Y F444 1.40E−07P257A/T307A/Q311A/M428L/N434Y F445 6.20E−08P257A/T307Q/Q311H/M428L/N434Y F446 1.10E−07P257A/T307A/Q311H/M428L/N434Y F447 1.40E−08P257A/N286E/T307Q/M428L/N434Y F448 5.30E−08P257A/N286E/T307A/M428L/N434Y F449 5.70E−07S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y F450 5.20E−07S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y F451 1.00E−07P257S/T307A/M428L/N434Y F452 1.40E−07 P257M/T307A/M428L/N434Y F4537.80E−08 P257N/T307A/M428L/N434Y F454 9.60E−08 P257I/T307A/M428L/N434YF455 2.70E−08 P257V/T307Q/M428Y/N434Y F456 3.40E−08P257V/T307Q/M428F/N434Y F457 4.00E−08 S239K/P257V/V308P/M428L/N434Y F4581.50E−08 P257V/T307Q/V308P/N325G/M428L/N434Y F459 1.30E−08P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y F460 4.70E−08P257V/T307A/V308P/N325G/M428L/N434Y F462 8.50E−08P257A/V308P/N325G/M428L/N434Y F463 1.30E−07P257A/T307A/V308P/M428L/N434Y F464 5.50E−08P257A/T307Q/V308P/M428L/N434Y F465 2.10E−08P257V/N286E/T307Q/N325G/M428L/N434Y F466 3.50E−07 T256E/P257V/N434Y F4675.70E−07 T256E/P257T/N434Y F468 5.70E−08 S239K/P257T/V308P/M428L/N434YF469 5.60E−08 P257T/V308P/N325G/M428L/N434Y F470 5.40E−08T256E/P257T/V308P/N325G/M428L/N434Y F471 6.60E−08P257T/V308P/N325G/E382A/M428L/N434Y F472 5.40E−08P257T/V308P/N325G/P387E/M428L/N434Y F473 4.50E−07P257T/V308P/L309P/N325G/M428L/N434Y F474 3.50E−07P257T/V308P/L309R/N325G/M428L/N434Y F475 4.30E−08T256E/P257V/T307Q/M428L/N434Y F476 5.50E−08P257V/T307Q/E382A/M428L/N434Y F477 4.30E−08P257V/T307Q/P387E/M428L/N434Y F480 3.90E−08 P257L/V308P/N434Y F4815.60E−08 P257T/T307Q/N434Y F482 7.00E−08 P257V/T307Q/N325G/N434Y F4835.70E−08 P257V/T307Q/Q311A/N434Y F484 6.20E−08 P257V/V305A/T307Q/N434YF485 9.70E−08 P257V/N286E/T307A/N434Y F486 3.40E−07P257V/T307Q/L309R/Q311H/M428L/N434Y F488 3.50E−08P257V/V308P/N325G/M428L/N434Y F490 7.50E−08S239K/P257V/V308P/Q311H/M428L/N434Y F492 9.80E−08P257V/V305A/T307A/N325G/M428L/N434Y F493 4.90E−07S239K/D270F/T307P/N325G/M428Y/N434Y F497 3.10E−06P257T/T307A/M428V/N434Y F498 1.30E−06 P257A/M428V/N434Y F499 5.20E−07P257A/T307A/M428V/N434Y F500 4.30E−08 P257S/T307Q/M428L/N434Y F5061.90E−07 P257V/N297A/T307Q/M428L/N434Y

Table 13-8 is the continuation of Table 13-7.

TABLE 13-8 F507 5.10E−08 P257V/N286A/T307Q/M428L/N434Y F508 1.10E−07P257V/T307Q/N315A/M428L/N434Y F509 5.80E−08P257V/T307Q/N384A/M428L/N434Y F510 5.30E−08P257V/T307Q/N389A/M428L/N434Y F511 4.20E−07 P257V/N434Y F512 5.80E−07P257T/N434Y F517 3.10E−07 P257V/N286E/N434Y F518 4.20E−07P257T/N286E/N434Y F519 2.60E−08 P257V/N286E/T307Q/N434Y F521 1.10E−08P257V/N286E/T307Q/M428Y/N434Y F523 2.60E−08P257V/V305A/T307Q/M428Y/N434Y F526 1.90E−08 P257T/T307Q/M428Y/N434Y F5279.40E−09 P257V/T307Q/V308P/N325G/M428Y/N434Y F529 2.50E−08P257T/T307Q/M428F/N434Y F533 1.20E−08 P257A/N286E/T307Q/M428F/N434Y F5341.20E−08 P257A/N286E/T307Q/M428Y/N434Y F535 3.90E−08T250A/P257V/T307Q/M428L/N434Y F538 9.90E−08T250F/P257V/T307Q/M428L/N434Y F541 6.00E−08T250I/P257V/T307Q/M428L/N434Y F544 3.10E−08T250M/P257V/T307Q/M428L/N434Y F549 5.40E−08T250S/P257V/T307Q/M428L/N434Y F550 5.90E−08T250V/P257V/T307Q/M428L/N434Y F551 1.20E−07T250W/P257V/T307Q/M428L/N434Y F552 1.10E−07T250Y/P257V/T307Q/M428L/N434Y F553 1.70E−07 M252Y/Q311A/N434Y F5542.80E−08 S239K/M252Y/S254T/V308P/N434Y F556 1.50E−06 M252Y/T307Q/Q311AF559 8.00E−08 M252Y/S254T/N286E/N434Y F560 2.80E−08M252Y/S254T/V308P/N434Y F561 1.40E−07 M252Y/S254T/T307A/N434Y F5628.30E−08 M252Y/S254T/T307Q/N434Y F563 1.30E−07 M252Y/S254T/Q311A/N434YF564 1.90E−07 M252Y/S254T/Q311H/N434Y F565 9.20E−08M252Y/S254T/T307A/Q311A/N434Y F566 6.10E−08M252Y/S254T/T307Q/Q311A/N434Y F567 2.20E−07 M252Y/S254T/M428I/N434Y F5681.10E−07 M252Y/T256E/T307A/Q311H/N434Y F569 2.00E−07M252Y/T256Q/T307A/Q311H/N434Y F570 1.30E−07M252Y/S254T/T307A/Q311H/N434Y F571 8.10E−08M252Y/N286E/T307A/Q311H/N434Y F572 1.00E−07M252Y/T307A/Q311H/M428I/N434Y F576 1.60E−06 M252Y/T256E/T307Q/Q311H F5771.30E−06 M252Y/N286E/T307A/Q311A F578 5.70E−07 M252Y/N286E/T307Q/Q311AF580 8.60E−07 M252Y/N286E/T307Q/Q311H F581 7.20E−08M252Y/T256E/N286E/N434Y F582 7.50E−07 S239K/M252Y/V308P F583 7.80E−07S239K/M252Y/V308P/E382A F584 6.30E−07 S239K/M252Y/T256E/V308P F5852.90E−07 S239K/M252Y/N286E/V308P F586 1.40E−07S239K/M252Y/N286E/V308P/M428I F587 1.90E−07 M252Y/N286E/M428L/N434Y F5922.00E−07 M252Y/S254T/E382A/N434Y F593 3.10E−08S239K/M252Y/S254T/V308P/M428I/N434Y F594 1.60E−08S239K/M252Y/T256E/V308P/M428I/N434Y F595 1.80E−07S239K/M252Y/M428I/N434Y F596 4.00E−07 M252Y/D312A/E382A/M428Y/N434Y F5972.20E−07 M252Y/E382A/P387E/N434Y F598 1.40E−07 M252Y/D312A/P387E/N434YF599 5.20E−07 M252Y/P387E/M428Y/N434Y

Table 13-9 is the continuation of Table 13-8.

TABLE 13-9 F600 2.80E−07 M252Y/T256Q/E382A/N434Y F601 9.60E−09M252Y/N286E/V308P/N434Y F608 G236A/S239D/I332E F611 2.80E−07M252Y/V305T/T307P/V308I/L309A/N434Y F612 3.60E−07M252Y/T307P/V308I/L309A/N434Y F613 S239D/A330L/I332E F616S239D/K326D/L328Y F617 7.40E−07 S239K/N434W F618 6.40E−07S239K/V308F/N434Y F619 3.10E−07 S239K/M252Y/N434Y F620 2.10E−07S239K/M252Y/S254T/N434Y F621 1.50E−07 S239K/M252Y/T307A/Q311H/N434Y F6223.50E−07 S239K/M252Y/T256Q/N434Y F623 1.80E−07 S239K/M252W/N434W F6241.40E−08 S239K/P257A/N286E/T307Q/M428L/N434Y F625 7.60E−08S239K/P257A/T307Q/M428L/N434Y F626 1.30E−06 V308P F629 3.90E−08M252Y/V279L/V308P/N434Y F630 3.70E−08 S239K/M252Y/V279L/V308P/N434Y F6332.40E−08 M252Y/V282D/V308P/N434Y F634 3.20E−08S239K/M252Y/V282D/V308P/N434Y F635 4.50E−08 M252Y/V284K/V308P/N434Y F6364.80E−08 S239K/M252Y/V284K/V308P/N434Y F637 1.50E−07M252Y/K288S/V308P/N434Y F638 1.40E−07 S239K/M252Y/K288S/V308P/N434Y F6392.70E−08 M252Y/V308P/G385R/N434Y F640 3.60E−08S239K/M252Y/V308P/G385R/N434Y F641 3.00E−08 M252Y/V308P/Q386K/N434Y F6423.00E−08 S239K/M252Y/V308P/Q386K/N434Y F643 3.20E−08L235G/G236R/S239K/M252Y/V308P/N434Y F644 3.00E−08G236R/S239K/M252Y/V308P/N434Y F645 3.30E−08S239K/M252Y/V308P/L328R/N434Y F646 3.80E−08S239K/M252Y/N297A/V308P/N434Y F647 2.90E−08 P238D/M252Y/V308P/N434Y F648P238D F649 1.20E−07 S239K/M252Y/N286E/N434Y F650 1.70E−07S239K/M252Y/T256E/N434Y F651 1.80E−07 S239K/M252Y/Q311A/N434Y F6522.40E−07 P238D/M252Y/N434Y F654 3.20E−08 L235K/S239K/M252Y/V308P/N434YF655 3.40E−08 L235R/S239K/M252Y/V308P/N434Y F656 3.30E−08G237K/S239K/M252Y/V308P/N434Y F657 3.20E−08G237R/S239K/M252Y/V308P/N434Y F658 3.20E−08P238K/S239K/M252Y/V308P/N434Y F659 3.00E−08P238R/S239K/M252Y/V308P/N434Y F660 3.10E−08S239K/M252Y/V308P/P329K/N434Y F661 3.40E−08S239K/M252Y/V308P/P329R/N434Y F663 6.40E−09S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y F664 3.90E−08M252Y/N286A/V308P/N434Y F665 2.00E−08 M252Y/N286D/V308P/N434Y F6662.10E−08 M252Y/N286F/V308P/N434Y F667 3.00E−08 M252Y/N286G/V308P/N434YF668 4.00E−08 M252Y/N286H/V308P/N434Y F669 3.50E−08M252Y/N286I/V308P/N434Y F670 2.10E−07 M252Y/N286K/V308P/N434Y F6712.20E−08 M252Y/N286L/V308P/N434Y F672 2.40E−08 M252Y/N286M/V308P/N434YF673 2.30E−08 M252Y/N286P/V308P/N434Y F674 3.20E−08M252Y/N286Q/V308P/N434Y F675 5.10E−08 M252Y/N286R/V308P/N434Y

Table 13-10 is the continuation of Table 13-9.

TABLE 13-10 F676 3.20E−08 M252Y/N286S/V308P/N434Y F677 4.70E−08M252Y/N286T/V308P/N434Y F678 3.30E−08 M252Y/N286V/V308P/N434Y F6791.70E−08 M252Y/N286W/V308P/N434Y F680 1.50E−08 M252Y/N286Y/V308P/N434YF681 4.90E−08 M252Y/K288A/V308P/N434Y F682 8.20E−08M252Y/K288D/V308P/N434Y F683 5.00E−08 M252Y/K288E/V308P/N434Y F6845.10E−08 M252Y/K288F/V308P/N434Y F685 5.30E−08 M252Y/K288G/V308P/N434YF686 4.60E−08 M252Y/K288H/V308P/N434Y F687 4.90E−08M252Y/K288I/V308P/N434Y F688 2.80E−08 M252Y/K288L/V308P/N434Y F6894.10E−08 M252Y/K288M/V308P/N434Y F690 1.00E−07 M252Y/K288N/V308P/N434YF691 3.20E−07 M252Y/K288P/V308P/N434Y F692 3.90E−08M252Y/K288Q/V308P/N434Y F693 3.60E−08 M252Y/K288R/V308P/N434Y F6944.70E−08 M252Y/K288V/V308P/N434Y F695 4.00E−08 M252Y/K288W/V308P/N434YF696 4.40E−08 M252Y/K288Y/V308P/N434Y F697 3.10E−08S239K/M252Y/V308P/N325G/N434Y F698 2.20E−08M252Y/N286E/T307Q/Q311A/N434Y F699 2.30E−08S239K/M252Y/N286E/T307Q/Q311A/N434Y F700 5.20E−08M252Y/V308P/L328E/N434Y F705 7.10E−09 M252Y/N286E/V308P/M428I/N434Y F7061.80E−08 M252Y/N286E/T307Q/Q311A/M428I/N434Y F707 5.90E−09M252Y/N286E/T307Q/V308P/Q311A/N434Y F708 4.10E−09M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y F709 2.00E−08S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y F710 1.50E−08P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y F711 6.50E−08S239K/M252Y/T307Q/Q311A/N434Y F712 6.00E−08P238D/M252Y/T307Q/Q311A/N434Y F713 2.00E−08P238D/M252Y/N286E/T307Q/Q311A/N434Y F714 2.30E−07P238D/M252Y/N325S/N434Y F715 2.30E−07 P238D/M252Y/N325M/N434Y F7162.70E−07 P238D/M252Y/N325L/N434Y F717 2.60E−07 P238D/M252Y/N325I/N434YF718 2.80E−07 P238D/M252Y/Q295M/N434Y F719 7.40E−08P238D/M252Y/N325G/N434Y F720 2.40E−08 M252Y/T307Q/V308P/Q311A/N434Y F7211.50E−08 M252Y/T307Q/V308P/Q311A/M428I/N434Y F722 2.70E−07P238D/M252Y/A327G/N434Y F723 2.80E−07 P238D/M252Y/L328D/N434Y F7242.50E−07 P238D/M252Y/L328E/N434Y F725 4.20E−08L235K/G237R/S239K/M252Y/V308P/N434Y F726 3.70E−08L235K/P238K/S239K/M252Y/V308P/N434Y F729 9.20E−07 T307A/Q311A/N434Y F7306.00E−07 T307Q/Q311A/N434Y F731 8.50E−07 T307A/Q311H/N434Y F732 6.80E−07T307Q/Q311H/N434Y F733 3.20E−07 M252Y/L328E/N434Y F734 3.10E−07G236D/M252Y/L328E/N434Y F736 3.10E−07 M252Y/S267M/L328E/N434Y F7373.10E−07 M252Y/S267L/L328E/N434Y F738 3.50E−07 P238D/M252Y/T307P/N434YF739 2.20E−07 M252Y/T307P/Q311A/N434Y F740 2.90E−07M252Y/T307P/Q311H/N434Y F741 3.10E−07 P238D/T250A/M252Y/N434Y F7449.90E−07 P238D/T250F/M252Y/N434Y

Table 13-11 is the continuation of Table 13-10.

TABLE 13-11 F745 6.60E−07 P238D/T250G/M252Y/N434Y F746 6.00E−07P238D/T250H/M252Y/N434Y F747 2.80E−07 P238D/T250I/M252Y/N434Y F7495.10E−07 P238D/T250L/M252Y/N434Y F750 3,00E−07 P238D/T250M/M252Y/N434YF751 5.30E−07 P238D/T250N/M252Y/N434Y F753 1.80E−07P238D/T250Q/M252Y/N434Y F755 3.50E−07 P238D/T250S/M252Y/N434Y F7563.70E−07 P238D/T250V/M252Y/N434Y F757 1.20E−06 P238D/T250W/M252Y/N434YF758 1.40E−06 P238D/T250Y/M252Y/N434Y F759 L235K/S239K F760 L235R/S239KF761 1.10E−06 P238D/N434Y F762 3.60E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y F763 3.50E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y F764 6.30E−07P238D/T307Q/Q311A/N434Y F765 8.50E−08P238D/M252Y/T307Q/L309E/Q311A/N434Y F766 6.00E−07T307A/L309E/Q311A/N434Y F767 4.30E−07 T307Q/L309E/Q311A/N434Y F7686.40E−07 T307A/L309E/Q311H/N434Y F769 4.60E−07 T307Q/L309E/Q311H/N434YF770 3.00E−07 M252Y/T256A/N434Y F771 4.00E−07 M252Y/E272A/N434Y F7723.80E−07 M252Y/K274A/N434Y F773 3.90E−07 M252Y/V282A/N434Y F774 4.00E−07M252Y/N286A/N434Y F775 6.20E−07 M252Y/K338A/N434Y F776 3.90E−07M252Y/K340A/N434Y F777 3.90E−07 M252Y/E345A/N434Y F779 3.90E−07M252Y/N361A/N434Y F780 3.90E−07 M252Y/Q362A/N434Y F781 3.70E−07M252Y/S375A/N434Y F782 3.50E−07 M252Y/Y391A/N434Y F783 4.00E−07M252Y/D413A/N434Y F784 5.00E−07 M252Y/L309A/N434Y F785 7.40E−07M252Y/L309H/N434Y F786 2.80E−08 M252Y/S254T/N286E/T307Q/Q311A/N434Y F7878.80E−08 M252Y/S254T/T307Q/L309E/Q311A/N434Y F788 4.10E−07M252Y/N315A/N434Y F789 1.50E−07 M252Y/N315D/N434Y F790 2.70E−07M252Y/N315E/N434Y F791 4.40E−07 M252Y/N315F/N434Y F792 4.40E−07M252Y/N315G/N434Y F793 3.30E−07 M252Y/N315I/N434Y F794 4.10E−07M252Y/N315K/N434Y F795 3.10E−07 M252Y/N315L/N434Y F796 3.40E−07M252Y/N315M/N434Y F798 3.50E−07 M252Y/N315Q/N434Y F799 4.10E−07M252Y/N315R/N434Y F800 3.80E−07 M252Y/N315S/N434Y F801 4.40E−07M252Y/N315T/N434Y F802 3.30E−07 M252Y/N315V/N434Y F803 3.60E−07M252Y/N315W/N434Y F804 4.00E−07 M252Y/N315Y/N434Y F805 3.00E−07M252Y/N325A/N434Y F806 3.10E−07 M252Y/N384A/N434Y F807 3.20E−07M252Y/N389A/N434Y F808 3.20E−07 M252Y/N389A/N390A/N434Y F809 2.20E−07M252Y/S254T/T256S/N434Y

Table 13-12 is the continuation of Table 13-11.

TABLE 13-12 F810 2.20E−07 M252Y/A378V/N434Y F811 4.90E−07M252Y/E380S/N434Y F812 2.70E−07 M252Y/E382V/N434Y F813 2.80E−07M252Y/S424E/N434Y F814 1.20E−07 M252Y/N434Y/Y436I F815 5.50E−07M252Y/N434Y/T437R F816 3.80E−07 P238D/T250V/M252Y/T307P/N434Y F8179.80E−08 P238D/T250V/M252Y/T307Q/Q311A/N434Y F819 1.40E−07P238D/M252Y/N286E/N434Y F820 3.40E−07 L235K/S239K/M252Y/N434Y F8213.10E−07 L235R/S239K/M252Y/N434Y F822 1.10E−06P238D/T250Y/M252Y/W313Y/N434Y F823 1.10E−06P238D/T250Y/M252Y/W313F/N434Y F828 2.50E−06P238D/T250V/M252Y/I253V/N434Y F831 1.60E−06P238D/T250V/M252Y/R255A/N434Y F832 2.60E−06P238D/T250V/M252Y/R255D/N434Y F833 8.00E−07P238D/T250V/M252Y/R255E/N434Y F834 8.10E−07P238D/T250V/M252Y/R255F/N434Y F836 5.00E−07P238D/T250V/M252Y/R255H/N434Y F837 5.60E−07P238D/T250V/M252Y/R255I/N434Y F838 4.30E−07P238D/T250V/M252Y/R255K/N434Y F839 3.40E−07P238D/T250V/M252Y/R255L/N434Y F840 4.20E−07P238D/T250V/M252Y/R255M/N434Y F841 1.10E−06P238D/T250V/M252Y/R255N/N434Y F843 6.60E−07P238D/T250V/M252Y/R255Q/N434Y F844 1.30E−06P238D/T250V/M252Y/R255S/N434Y F847 3.40E−07P238D/T250V/M252Y/R255W/N434Y F848 8.30E−07P238D/T250V/M252Y/R255Y/N434Y F849 3.30E−07 M252Y/D280A/N434Y F8502.90E−07 M252Y/D280E/N434Y F852 3.30E−07 M252Y/D280G/N434Y F853 3.20E−07M252Y/D280H/N434Y F855 3.20E−07 M252Y/D280K/N434Y F858 3.20E−07M252Y/D280N/N434Y F860 3.30E−07 M252Y/D280Q/N434Y F861 3.20E−07M252Y/D280R/N434Y F862 3.00E−07 M252Y/D280S/N434Y F863 2.70E−07M252Y/D280T/N434Y F867 2.80E−07 M252Y/N384A/N389A/N434Y F868 2.00E−08G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y F869 G236A/S239D F870 7.30E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y F871 7.10E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y F872 1.30E−07L235K/S239K/M252Y/N286E/N434Y F873 1.20E−07L235R/S239K/M252Y/N286E/N434Y F875 4.80E−07 M252Y/N434Y/Y436A F8778.30E−07 M252Y/N434Y/Y436E F878 1.90E−07 M252Y/N434Y/Y436F F879 9.20E−07M252Y/N434Y/Y436G F880 3.90E−07 M252Y/N434Y/Y436H F881 3.10E−07M252Y/N434Y/Y436K F882 1.30E−07 M252Y/N434Y/Y436L F883 2.10E−07M252Y/N434Y/Y436M F884 4.00E−07 M252Y/N434Y/Y436N F888 4.80E−07M252Y/N434Y/Y436S F889 2.20E−07 M252Y/N434Y/Y436T F890 1.10E−07M252Y/N434Y/Y436V F891 1.70E−07 M252Y/N434Y/Y436W F892 7.10E−08M252Y/S254T/N434Y/Y436I F893 9.80E−08 L235K/S239K/M252Y/N434Y/Y436I

Table 13-13 is the continuation of Table 13-12.

TABLE 13-13 F894 9.20E−08 L235R/S239K/M252Y/N434Y/Y436I F895 2.10E−08L235K/8239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F896 2.00E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F897 9.70E−08M252Y/N315D/N384A/N389A/N434Y F898 1.70E−07M252Y/N315E/N384A/N389A/N434Y F899 1.10E−07 M252Y/N315D/G316A/N434Y F9001.70E−07 M252Y/N315D/G316D/N434Y F901 1.30E−07 M252Y/N315D/G316E/N434YF902 2.20E−07 M252Y/N315D/G316F/N434Y F903 2.30E−07M252Y/N315D/G316H/N434Y F904 1.00E−07 M252Y/N315D/G316I/N434Y F9051.30E−07 M252Y/N315D/G316K/N434Y F906 1.50E−07 M252Y/N315D/G316L/N434YF907 1.30E−07 M252Y/N315D/G316M/N434Y F908 1.50E−07M252Y/N315D/G316N/N434Y F909 1.30E−07 M252Y/N315D/G316P/N434v F9101.40E−07 M252Y/N315D/G316Q/N434Y F911 1.30E−07 M252Y/N315D/G316R/N434YF912 1.20E−07 M252Y/N315D/G316S/N1434Y F913 1.10E−07M252Y/N315D/G316T/N434Y F914 1.60E−07 M252Y/N315D/G316V/N434Y F9152.30E−07 M252Y/N315D/G316W/N434Y F917 2.50E−07 M252Y/N286S/N434Y F9182.80E−07 M252Y/D280E/N384A/N389A/N434Y F919 3.30E−07M252Y/D280G/N384A/N389A/N434Y F920 2.50E−07M252Y/N286S/N384A/N389A/N434Y F921 1.20E−07M252Y/N286E/N384A/N389A/N434Y F922 5.90E−08L235K/S239K/M252Y/N286E/N434Y/Y436I F923 6.00E−08L235R/S239K/M252Y/N286E/N434Y/Y436I F924 3.40E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F925 3.20E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F926 1.10E−07L235K/S239K/M252Y/S254T/N434Y/Y436I F927 1.00E−07L235R/S239K/M252Y/S254T/N434Y/Y436I F928 2.90E−08M252Y/T307Q/Q311A/N434Y/Y436I F929 2.90E−08M252Y/S254T/T307Q/Q311A/N434Y/Y436I F930 1.40E−07P238D/T250V/M252Y/N286E/N434Y F931 1.20E−07P238D/T250V/M252Y/N434Y/Y436I F932 3.20E−07 T250V/M252Y/N434Y F9333.00E−07 L234R/P238D/M250V/M252Y/N434Y F934 3.10E−07G236K/P238D/T250V/M252Y/N434Y F935 3.20E−07G237K/P238D/T250V/M252Y/N434Y F936 3.20E−07G237R/P238D/T250V/M252Y/N434Y F937 3.10E−07P238D/S239K/T250V/M252Y/N434Y F938 1.60E−07L235K/S239K/M252Y/N434Y/Y436V F939 1.50E−07L235R/S239K/M252Y/N434Y/Y436V F940 1.50E−07P238D/T250V/M252Y/N434Y/Y436V F941 1.20E−08M252Y/N286E/T307Q/Q311A/N434Y/Y436V F942 4.20E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F943 4.00E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F944 1.70E−07T250V/M252Y/N434Y/Y436V F945 1.70E−08 T250V/M252Y/V308P/N434Y/Y436V F9464.30E−08 T250V/M252Y/T307Q/Q311A/N434Y/Y436V F947 1.10E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F954 5.30E−07M252Y/N434Y/H435K/Y436V F957 7.70E−07 M252Y/N434Y/H435N/Y436V F9608.00E−07 M252Y/N434Y/H435R/Y436V F966 3.10E−07 M252Y/S254A/N434Y F9702.50E−06 M252Y/S254G/N434Y F971 2.60E−06 M252Y/S254H/N434Y F972 2.60E−07M252Y/S254I/N434Y

Table 13-14 is the continuation of Table 13-13.

TABLE 13-14 F978 1.30E−06 M252Y/S254Q/N434Y F980 1.80E−07M252Y/S254V/N434Y F987 4.00E−08P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F988 6.90E−08P238D/T250V/M252Y/N286E/N434Y/Y436V F989 1.40E−08L235R/8239K/M252Y/V308P/N434Y/Y436V F990 9.40E−09L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F991 1.30E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F992 5.10E−08L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F993 3.80E−08M252Y/T307Q/Q311A/N434Y/Y436V F994 2.80E−07 M252Y/N325G/N434Y F9952.90E−07 L235R/P238D/S239K/M252Y/N434Y F996 1.30E−07L235R/P238D/S239K/M252Y/N434Y/Y436V F997 3.80E−07K248I/T250V/M252Y/N434Y/Y436V F998 8.50E−07K248Y/T250V/M252Y/N434Y/Y436V F999 2.10E−07T250V/M252Y/E258H/N434Y/Y436V F1005 N325G F1008 1.70E−07L235R/S239K/T250V/M252Y/N434Y/Y436V F1009 1.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1010 1.90E−07L235R/S239K/M252Y/T307A/Q311H/N434Y F1011 4.50E−08T250V/M252Y/V308P/N434Y F1012 4.70E−08L235R/S239K/T250V/M252Y/V308P/N434Y F1013 3.00E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y F1014 3.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1015 2.20E−08L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1016 3.80E−09T250V/M252Y/N286E/T307Q/V308F/Q311A/N434Y/Y436V F1017 4.20E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10183.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10193.40E−07 P238D/T250V/M252Y/N325G/N434Y F1020 8.50E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y F1021 3.30E−07P238D/T250V/M252Y/N325A/N434Y F1022 K326D/L328Y F1023 4.40E−08S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1024 4.00E−08T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F1025 3.60E−08S239D/T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F1026 8.40E−08M252Y/T307A/Q311H/N434Y/Y436V F1027 8.60E−08L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1028 4.60E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1029 5.10E−08T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1030 I332E F1031 5.30E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1032 4.30E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V F1033 1.00E−06P238D/N434W F1034 1.50E−08 L235K/S239K/M252Y/V308P/N434Y/Y436V F10351.00E−08 L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1036 1.40E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F1037 6.10E−08L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F1038 2.80E−07L235K/P238D/S239K/M252Y/N434Y F1039 1.30E−07L235K/P238D/S239K/M252Y/N434Y/Y436V F1040 2.00E−07L235K/S239K/T250V/M252Y/N434Y/Y436V F1041 1.40E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1042 2.00E−07L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.20E−08L235K/S239K/T250V/M252Y/V308P/N434Y F1044 3.50E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1045 2.50E−08L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1046 4.50E−09L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10473.40E−09 L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10489.90E−08 L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1050 3.50E−09T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F1051 3.90E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F10523.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N1428I/N434Y/Y436V

Reference Example 1 Construction of Antibody Expression Vectors; andExpression and Purification of Antibodies

Synthesis of full-length genes encoding the nucleotide sequences of theH chain and L chain of the antibody variable regions was carried out byproduction methods known to those skilled in the art using Assemble PCRand such. Introduction of amino acid substitutions was carried out bymethods known to those skilled in the art using PCR or such. Theobtained plasmid fragment was inserted into an animal cell expressionvector, and the H-chain expression vector and L-chain expression vectorwere produced. The nucleotide sequence of the obtained expression vectorwas determined by methods known to those skilled in the art. Theproduced plasmids were introduced transiently into the HEK293H cell linederived from human embryonic kidney cancer cells (Invitrogen) or intoFreeStyle293 cells (Invitrogen) for antibody expression. The obtainedculture supernatant was collected, and then passed through a 0.22 μmMILLEX(R)-GV filter (Millipore), or through a 0.45 μm MILLEX(R)-GVfilter (Millipore) to obtain the culture supernatant. Antibodies werepurified from the obtained culture supernatant by methods known to thoseskilled in the art using rProtein A Sepharose Fast Flow (GE Healthcare)or Protein G Sepharose 4 Fast Flow (GE Healthcare). For theconcentration of the purified antibodies, their absorbance at 280 nm wasmeasured using a spectrophotometer. From the obtained value, theextinction coefficient calculated by the methods such as PACE was usedto calculate the antibody concentration (Protein Science 1995; 4:2411-2423).

Reference Example 2 Method for Preparing FcγR and Method for Analyzingthe Interaction Between an Altered Antibody and FcγR

Extracellular domains of FcγRs were prepared by the following method.First, a gene of the extracellular domain of FcγR was synthesized by amethod well known to those skilled in the art. At that time, thesequence of each FcγR was produced based on the information registeredat NCBI. Specifically, FcγRI was produced based on the sequence of NCBIAccession No. NM_(—)000566 (Version No. NM_(—)000566.3), FcγRIIa wasproduced based on the sequence of NCBI Accession No. NM_(—)001136219(Version No. NM_(—)001136219.1), FcγRIIb was produced based on thesequence of NCBI Accession No. NM_(—)004001 (Version No.NM_(—)004001.3), FcγRIIIa was produced based on the sequence of NCBIAccession No. NM_(—)001127593 (Version No. NM_(—)001127593.1), andFcγRIIIb was produced based on the sequence of NCBI Accession No.NM_(—)000570 (Version No. NM_(—)000570.3), and a His tag was attached tothe C terminus. Furthermore, the presence of polymorphism is known forFcγRIIa, FcγRIIIa, and FcγRIIIb, and the polymorphic sites were producedby referring to Warmerdam et al. (J. Exp. Med., 1990, 172: 19-25) forFcγRIIa; Wu et al. (J. Clin. Invest., 1997, 100 (5): 1059-1070) forFcγRIIIa; and Ory et al. (J. Clin. Invest., 1989, 84, 1688-1691) forFcγRIIIb.

The obtained gene fragments were inserted into an animal cell expressionvector, and expression vectors were produced. The produced expressionvectors were introduced transiently into human embryonic kidney cancercell-derived FreeStyle293 cells (Invitrogen) to express the proteins ofinterest. Regarding FcγRIIb used for crystal structure analysis, theprotein of interest was expressed in the presence of Kifunesine at afinal concentration of 10 μg/mL, so that the sugar chain added toFcγRIIb will be the high-mannose type. Cells were cultured, and aftercollection of the obtained culture supernatant, this was passed througha 0.22 μm filter to obtain the culture supernatant. In principle, theobtained culture supernatants were purified in the following four steps.The steps carried out were, cation exchange column chromatography (SPSepharose FF) in step 1, affinity column chromatography (HisTrap HP) forHis tag in step 2, gel filtration column chromatography (Superdex200) instep 3, and aseptic filtration in step 4. However, for FcγRI, anionexchange column chromatography using Q sepharose FF was performed asstep 1. The purified proteins were subjected to absorbance measurementsat 280 nm using a spectrophotometer; and from the obtained values, theconcentrations of the purified proteins were calculated using theabsorption coefficient calculated using methods such as PACE (ProteinScience 1995; 4: 2411-2423).

Analysis of interaction between each altered antibody and the Fcγreceptor prepared as mentioned above was carried out using Biacore T100(GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100, and Biacore4000. HBS-EP+ (GE Healthcare) was used as the running buffer, and themeasurement temperature was set to 25° C. Chips produced by immobilizingthe antigen peptide, Protein A (Thermo Scientific), Protein A/G (ThermoScientific), and Protein L (ACTIGEN or BioVision) by the amine couplingmethod to a Series S sensor Chip CM5 (GE Healthcare) or Series S sensorChip CM4 (GE Healthcare), or alternatively, chips produced by allowingpreliminarily biotinylated antigen peptides to interact with andimmobilize onto a Series S Sensor Chip SA (certified) (GE Healthcare)were used.

After capturing of antibodies of interest onto these sensor chips, anFcγ receptor diluted with the running buffer was allowed to interact,the amount bound to an antibody was measured, and compared among theantibodies. However, since the amount of Fcγ receptor bound depends onthe amount of the captured antibodies, the amount of Fcγ receptor boundwas divided by the amount of each antibody captured to obtain correctedvalues, and these values were compared. Furthermore, antibodies capturedonto the sensor chips were washed by reaction with 10 mM glycine-HCl, pH1.5, and the chips were regenerated and used repeatedly.

Kinetic analyses for calculating the KD values of each altered antibodyfor FcγR were performed according to the following method. First,antibodies of interest were captured onto the above-mentioned sensorchips, and an Fcγ receptor diluted with the running buffer was allowedto interact. The Biacore Evaluation Software was used to globally fitthe measured results regarding the obtained sensorgram using the 1:1Langmuir binding model, and the association rate constant ka (L/mol/s)and the dissociation rate constant kd (1/s) were calculated; and fromthose values the dissociation constants KD (mol/L) were calculated.

When the interaction between each of the altered antibodies and FcγR wasweak, and correct analysis was determined to be impossible by theabove-mentioned kinetic analysis, the KD for such interactions werecalculated using the following 1:1 binding model equation described inthe Biacore T100 Software Handbook BR1006-48 Edition AE.

The behavior of interacting molecules according to the 1:1 binding modelon Biacore can be described by Equation 1 shown below.

R _(eq) =C·R _(max)/(KD+C)+RI[Equation 1]

Req: a plot of steady state binding levels against analyte concentrationC: concentrationRI: bulk refractive index contribution in the sampleRmax: analyte binding capacity of the surface

When this equation is rearranged, KD can be expressed as Equation 2shown below.

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

KD can be calculated by substituting the values of Rmax, RI, and C intothis equation. The values of RI and C can be determined from thesensorgram of the measurement results and measurement conditions. Rmaxwas calculated according to the following method. As a target ofcomparison, for antibodies that had sufficiently strong interactions asevaluated simultaneously in the same round of measurement, the Rmaxvalue was obtained through global fitting using the 1:1 Langmuir bindingmodel, and then it was divided by the amount of the comparison antibodycaptured onto the sensor chip, and multiplied by the captured amount ofan altered antibody to be evaluated.

Reference Example 3 Preparation of Soluble Human IL-6 Receptor (hsIL-6R)

Recombinant human IL-6 receptor which is an antigen was prepared in themanner described below. A CHO line that constantly expresses solublehuman IL-6 receptor composed of an amino acid sequence consisting of the1st to 357th amino acid from the N terminus as reported in J. Immunol.(1994) 152, 4958-4968 (hereinafter referred to as hsIL-6R) wasconstructed using a method known among persons with ordinary skill inthe art. hsIL-6R was expressed by culturing this CHO line. hsIL-6R waspurified from culture supernatant of the resulting CHO line by the twosteps of Blue Sepharose 6 FF column chromatography and gel filtrationcolumn chromatography. The fraction that eluted as the main peak in thefinal step was used as the final purified product.

Reference Example 4 Elimination of Target Antigen from Plasma bypH-Dependent Binding Antibody Having Human FcRn-Binding Activity Under aNeutral pH Range Condition

When a conventional neutralizing antibody against a soluble antigen isadministered, the plasma retention of the antigen is expected to beprolonged by binding to the antibody. In general, antibodies have a longhalf-life (one week to three weeks) while the half-life of antigen isgenerally short (one day or less). Meanwhile, antibody-bound antigenshave a significantly longer half-life in plasma as compared to when theantigens are present alone. For this reason, administration of existingneutralizing antibody results in an increased antigen concentration inplasma. Such cases have been reported with various neutralizingantibodies that target soluble antigens including, for example, IL-6 (JImmunotoxicol. 2005, 3: 131-9), amyloid beta (mAbs, 2010, 2: 5, 1-13),MCP-1 (ARTHRITIS & RHEUMATISM 2006, 54: 2387-92), hepcidin (AAPS J.2010, 4, 646-57), and sIL-6 receptor (Blood. 2008 Nov. 15; 112(10):3959-64). Administration of existing neutralizing antibodies has beenreported to increase the total plasma antigen concentration to about 10to 1000 times (the level of increase varies depending on antigen) fromthe base line. Herein, the total plasma antigen concentration refers toa concentration as a total amount of antigen present in plasma, i.e.,the sum of concentrations of antibody-bound and antibody-unboundantigens. An increase in the total plasma antigen concentration isundesirable for such antibody pharmaceuticals that target a solubleantigen. The reason is that the plasma antibody concentration has to behigher than at least the total plasma antigen concentration toneutralize the soluble antigen. Specifically, “the total plasma antigenconcentration is increased to 10 to 1,000 times” means that, in order toneutralize the antigen, the plasma antibody concentration (i.e.,antibody dose) has to be 10 to 1,000 times higher as compared to whenincrease in the total plasma antigen concentration does not occur.Conversely, if the total plasma antigen concentration can be reduced by10 to 1,000 times as compared to the existing neutralizing antibody, theantibody dose can also be reduced to similar extent. Thus, antibodiescapable of decreasing the total plasma antigen concentration byeliminating the soluble antigen from plasma are highly useful ascompared to existing neutralizing antibodies.

The examination described in PCT/JP2011/001888 demonstrated thatantigen-binding molecules (IL-6 receptor-binding antibodies) withenhanced FcRn binding at pH 7.4 can reduce the total antigenconcentration in plasma by eliminating the soluble antigen, and theeffect to eliminate the soluble antigen is improved by conferring theproperty of binding to the antigen in a pH-dependent manner (binding tothe antigen at pH 7.4 in plasma and dissociating from the antigen at pH6.0 in the endosome). To reduce the total antigen concentration inplasma by administering an antigen-binding molecule, it is desirablethat the antigen-binding molecule comprises an antigen-binding domainand a human FcRn-binding domain, and the human FcRn-binding domain hashuman FcRn binding activity under an acidic condition and under aneutral condition, and the human FcRn binding activity is 3200 nM orgreater under a neutral condition. In this case, a controlantigen-binding molecule has the same antigen-binding domain, and itshuman FcRn-binding domain is a native human IgG Fc region.

FIG. 9 shows a mechanism in which soluble antigens are eliminated fromplasma by administering a pH-dependent antigen-binding antibody that hasincreased FcRn-binding activity at neutral pH as compared to aconventional neutralizing antibody. After binding to the soluble antigenin plasma, the existing neutralizing antibody that does not have thepH-dependent antigen-binding ability is slowly incorporated into cellsby non-specific interaction with the cells. The complex between theneutralizing antibody and soluble antigen incorporated into the cell istransferred to the acidic endosome and then recycled to plasma by FcRn.Meanwhile, the pH-dependent antigen-binding antibody that has theincreased FcRn-binding activity under the neutral condition is, afterbinding to the soluble antigen in plasma, rapidly incorporated intocells expressing FcRn on their cell membrane. Then, the soluble antigenbound to the pH-dependent antigen-binding antibody is dissociated fromthe antibody in the acidic endosome due to the pH-dependent bindingability. The soluble antigen dissociated from the antibody istransferred to the lysosome and degraded by proteolytic activity.Meanwhile, the antibody dissociated from the soluble antigen is recycledonto cell membrane by FcRn and then released to plasma again. The freeantibody, recycled as described above, can again bind to other solubleantigens. By repeating such cycle: FcRn-mediated uptake into cells;dissociation and degradation of the soluble antigen; and antibodyrecycling, such pH-dependent antigen-binding antibodies as describedabove having the increased FcRn binding activity under the neutralcondition can transfer a large amount of soluble antigen to the lysosomeand thereby decrease the total antigen concentration in plasma.

Reference Example 5 Demonstration of the In Vivo Pharmaceutical Effectof an Antibody without Neutralizing Activity In Vitro by EliminatingTarget Antigens from Plasma

It has been believed that, to exhibit the in vivo pharmaceutical effectto neutralize target antigens, antigen-binding molecules must have an invitro activity of neutralizing the target antigens. The reason is that,if ordinary antigen-binding molecules do not have in vitro neutralizingactivity, they cannot exhibit the in vivo pharmaceutical effect byneutralizing target antigens.

Meanwhile, Reference Example 4 shows that, when administered in vivo,pH-dependent binding antibodies that have human FcRn-binding activityunder a neutral pH range condition can eliminate target antigens fromplasma. The present inventors conceived that, if a target antigen waseliminated from plasma by administering an antibody, the action of thetarget antigen can be substantially blocked even when the antibody doesnot have antigen-neutralizing activity.

Isolation of the pH-Dependent Antibody 6RKE02-IgG1 Against Human IL-6Receptor from Human Naive Library

A human antibody phage display library consisting of multiple phagesthat display Fab domains of different human antibody sequences wasconstructed according to a method known to those skilled in the artusing as a template poly A RNA prepared from human PBMCs orcommercially-available human poly A RNA.

The first selection from the constructed naive human antibody phagedisplay library was performed by enriching only antibody fragments withantigen-binding ability. A biotin-labeled human IL-6 receptor was usedas an antigen.

Phages were produced by E. coli containing the constructed phage displayphagemids. To precipitate the phages, 2.5 M NaCl/10% PEG was added tothe E. coli culture media of phage production. The phages were dilutedwith TBS to prepare a phage library solution. Then, BSA and CaCl₂ wereadded at a final concentration of 4% BSA and a final calcium ionconcentration of 1.2 mM respectively to the phage library solution.Regarding the panning method, the present inventors referred to generalpanning methods using antigens immobilized onto magnetic beads (J.Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247(1-2), 191-203, Biotechnol. Prog. (2002) 18(2) 212-20, Mol. Cell.Proteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to theprepared phage library solution to contact it with the phage librarysolution at room temperature for 60 minutes. BSA-blocked magnetic beadswere added thereto and allowed to bind to antigen/phage complexes atroom temperature for 15 minutes. The beads were washed once with 1 ml of1.2 mM CaCl₂/TBS (TBS containing 1.2 mM CaCl₂). Then, a phage solutionwas collected according to a general method. The collected phagesolution was added to 10 ml of E. coli strain TG1 in the logarithmicgrowth phase (OD600 of 0.4-0.5). The E. coli was infected with thephages by culturing them while gently stirring at 37° C. for one hour.The infected E. coli was seeded in a 225 mm×225 mm plate. Then, thephages were collected from the culture medium of the seeded E. coli toprepare a phage library solution.

To enrich the phages, the second and subsequent pannings were performedusing the pH-dependent binding ability as an indicator. Specifically, 40pmol of the biotin-labeled antigen was added to the prepared phagelibrary solution, and contacted it with the phage library at roomtemperature for 60 minutes. BSA-blocked magnetic beads were addedthereto and allowed to bind to antigen/phage complexes at roomtemperature for 15 minutes. The beads were washed with 1 ml of 1.2 mMCaCl₂/TBST (TBS containing 1.2 mM CaCl₂ and 0.1% Tween 20) and with 1.2mM CaCl₂/TBS. Then, the beads combined with 0.1 ml of 50 mM MES/1.2 mMCaCl₂/150 mM NaCl (pH 5.5) were suspended at room temperature, andimmediately separated using a magnetic stand to collect a phagesolution. The collected phage solution was added to 10 ml of E. colistrain TG1 in the logarithmic growth phase (OD600 of 0.4-0.5). The E.coli was infected with the phages by culturing them while gentlystirring at 37° C. for one hour. The infected E. coli was seeded in a225 mm×225 mm plate. Then, the phages were collected from the culturemedium of the seeded E. coli to prepare a phage library solution. Thepanning using the pH-dependent binding ability as an indicator wasperformed repeatedly several times.

After repeating the panning twice, three times, or four times,phage-containing culture supernatants were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above.

To the phage-containing culture supernatants, BSA and CaCl₂ were addedat a final concentration of 4% BSA and at a final calcium ionconcentration of 1.2 mM, respectively. The phage-containing culturesupernatants were subjected to ELISA by the following procedure. AStreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μlof PBS containing the biotin-labeled antigen. After washing each well ofthe plate with PBST to remove the antigen, the wells were blocked with250 μl of 4% BSA/TBS for one hour or more. After removing 4% BSA/TBSfrom the wells, the culture supernatants prepared as mentioned abovewere added thereto. The antibodies presented on the phages were allowedto bind to the antigens on each well by incubating the plate at 37° C.for one hour. The wells were washed with 1.2 mM CaCl₂/TBST, and 1.2 mMCaCl₂/TBS (pH 7.6) or 1.2 mM CaCl₂/TBS (pH 5.5) was added thereto. Theplate was incubated by being allowed to stand at 37° C. for 30 minutes.After washing with 1.2 mM CaCl₂/TBST (pH 7.6), an HRP-linked anti-M13antibody (Amersham Pharmacia Biotech) diluted with TBS containing BSA ata final concentration of 4% and ionized calcium at a final concentrationof 1.2 mM was added to each well. The plate was incubated for one hour.After washing with 1.2 mM CaCl₂/TBST, TMB single solution (ZYMED) wasadded thereto. The chromogenic reaction in the solution of each well wasstopped by adding sulfuric acid, and then the absorbance at 450 nm wasmeasured to assess the color development.

Furthermore, the genes amplified with specific primers using as atemplate the clones subjected to phage ELISA were analyzed for theirnucleotide sequence.

Based on the results of the above-mentioned phage ELISA and sequenceanalysis, selection was performed using pooled libraries containing manyantibody fragments which were considered to have the ability to bind tothe antigen in a pH-dependent manner.

Expression of Antibodies

Antibody genes from pooled libraries containing many clones that wereconsidered to have the ability to bind to the antigen in a pH-dependentmanner based on the result of phage ELISA, were inserted into animalcell expression plasmids. Antibody expression was carried out using themethod described below. The FreeStyle 293-F line (Invitrogen) derivedfrom human fetal kidney cells were suspended in FreeStyle 293 ExpressionMedium (Invitrogen), and plated at a cell density of 2.63×10⁵ cells/mlin 190 μl to each well of a 96-well plate. The prepared plasmids wereintroduced into the cells by a lipofection method. The cells werecultured in a CO₂ incubator (37° C., 8% CO₂) for four days.

Analysis and Assessment of Interaction Using Biacore A100

Antibodies isolated by the above-described method were analyzed usingBiacore A100 (GE Healthcare) for the interaction between IL-6R and theantibodies of interest. The running buffer used was: 10 mM ACES, 150 mMNaCl, 1.2 mM CaCl₂, 0.05% Tween20, pH 7.4, or 10 mM ACES, 150 mM NaCl,1.2 mM CaCl₂, 0.05% Tween20, pH 6.0. The measurement temperature was 25°C. The chip used was a Series S Sencor Chip CM5 (GE Healthcare)immobilized with Protein A/G (Thermo Scientific) by an amine couplingmethod. Antibodies of interest were captured onto the chip, and allowedto interact with IL-6R diluted with the running buffer. The antibodiescaptured onto the chip were washed off by reacting 10 mM glycine-HCl (pH1.5), and the chip was regenerated for repeated use.

The IL-6R-binding activity of each antibody was assessed mainly using asan indicator the binding amount of IL-6R to the antibody. The amount ofchange (RU) in sensorgram upon interaction of the captured antibody withIL-6R, divided by the amount of change (RU) upon capturing the antibodyonto the chip, was used as the binding amount of IL-6R to the antibody.

Expression and Purification of Antibodies

Clones that were determined to have the ability to bind to the antigenin a pH-dependent manner, based on the result of screening withBiacoreA100, were expressed again to perform the assessment. Antibodieswere prepared using the method described in Reference Example 1.

Assessment of 6RKE02-IgG1 for its pH-Dependent Binding to Soluble HumanIL-6 Receptor

The above method yielded 6RKE02-IgG1 having 6RKE02H-IgG1 (SEQ ID NO: 1)as the heavy chain and 6RKE02L-k0 (SEQ ID NO: 2) as the light chain.6RKE02-IgG1 was analyzed for its interaction with IL-6R using BiacoreT100 (GE Healthcare), and the dissociation constant (KD) was calculated.

The running buffer used was 10 mM ACES (pH 7.4) containing 150 mM NaCland 0.05% Tween20. The measurement temperature was 37° C. The chip usedwas a Series S Sencor Chip CM4 (GE Healthcare) immobilized with ProteinA/G (Thermo Scientific) by an amine coupling method. Antibodies ofinterest were captured onto the chip, and allowed to interact with IL-6Rdiluted with the running buffer to 800, 400, 200, 100, 50, 25, and 12.5nM, and the running buffer at a flow rate of 2 μl/minute for 15 minutes.The antibodies captured onto the chip were washed off by reacting 10 mMglycine-HCl (pH 1.5), and the chip was regenerated for repeated use.

From the sensorgrams obtained as a result of the Biacore measurement,the dissociation constant KD (mol/l) of 6RKE02-IgG1 for IL-6R wascalculated by performing steady state affinity analysis using BiacoreEvaluation Software. The dissociation constant (KD) between 6RKE02-IgG1and IL-6R at pH 7.4, calculated by this method, was 1.4E-7 (M).

Next, the pH dependence of the binding of 6RKE02-IgG1 to hIL-6R wasassessed using Biacore T100. The running buffer used was: 10 mM ACES,150 mM NaCl, 0.05% Tween20, pH 7.4; and 10 mM ACES, 150 mM NaCl, 0.05%Tween20, pH 6.0. The measurement temperature was 37° C. The chip usedwas a Series S Sencor Chip CM4 (GE Healthcare) immobilized with ProteinA/G (Thermo Scientific) by an amine coupling method. Antibodies ofinterest were captured onto the chip, and allowed to interact withhIL-6R diluted with the running buffer to 1000, 250, and 62.5 nM, andthe running buffer.

Sensorgrams obtained by the measurement at pH 7.4 and pH 6.0 using thismethod are shown in FIG. 10. The captured amount of antibody has beennormalized to be 100 RU, and the binding phase and dissociation phase of6RKE02-IgG1 for hIL-6R are shown. A comparison of the results shown inFIG. 10 revealed that the binding of 6REK02-IgG1 to hIl-6R was reducedat pH 6.0 as compared to pH 7.4.

Assessment for Biological Activity Using BaF3 Cells Expressing HumanGp130 (BaF/gp130)

6RKE02-IgG1 and Tocilizumab were assessed for their IL-6receptor-neutralizing activity using BaF3/gp130 that shows humanIL-6/soluble human IL-6 receptor-dependent growth. After washing threetimes with RPMI1640 medium containing 10% FBS, BaF3/gp130 was suspendedin RPMI1640 medium containing 10% FBS and prepared at 1.5×10⁵ cells/mlwith a final concentration of 15 ng/ml for both human interleukin-6 (R&DSystems) and soluble human IL-6 receptor. This was aliquoted at 50 μl toeach well of a 96 well-plate (CORNING). Then, the purified antibodieswere serially diluted with PBS, and then diluted 20 times with RPMI1640medium containing 10% FBS, and added at 50 μl to each well. The cellswere cultured at 37° C. under 5% CO₂ for three days, and WST-8 reagent(Cell Counting Kit-8, DOJINDO LABORATORIES) diluted twice with PBS wasadded thereto at 20 μl/well. After four hours of incubation, theabsorbance at 450 nm (reference wavelength of 620 nm) was measured withmicroplate reader xMark (Bio-Rad Laboratories) to assess the IL-6receptor-neutralizing activity. The result is shown in FIG. 11.6RKE02-IgG1 did not inhibit the human IL-6/soluble human IL-6receptor-dependent growth of BaF3/gp130. Thus, it was shown that6RKE02-IgG1 does not have the neutralizing activity.

Preparation of 6RKE02-IgG1 with Increased FcRn-Binding Activity in aNeutral pH Range

To confer the mouse FcRn-binding activity under a neutral pH rangecondition to 6RKE02-IgG1, amino acid mutations were introduced into6RKE02H-IgG1, which is the heavy chain constant region of 6RKE02-IgG1.Specifically, 6RKE02H-F29 (SEQ ID NO: 3) was constructed by introducinginto 6RKE02H-IgG1 an amino acid substitution of Val for Ile at position332 (EU numbering) and an amino acid substitution of Tyr for Asn atposition 434 (EU numbering), using the method described in ReferenceExample 1. 6RKE02-F29, which contains 6RKE02H-F29 as the heavy chain and6RKE02L-k0 as the light chain, was constructed using the methoddescribed in Reference Example 1.

Kinetic Analysis for the Binding to Mouse FcRn

VH3/L(WT)-IgG1 comprising VH3-IgG1 (SEQ ID NO: 4) and L(WT)-CK (SEQ IDNO: 5), and VH3/L(WT)-F29 comprising VH3-F29 (SEQ ID NO: 6) and L(WT)-CK(SEQ ID NO: 5) were constructed to assess the mouse FcRn-bindingactivity of 6RKE02-F29.

Using VH3/L(WT)-IgG1 and VH3/L(WT)-F29, the mouse FcRn-binding activitywas assessed as follows.

Mouse FcRn and antibodies were kinetically analyzed using Biacore T100(GE Healthcare). An appropriate amount of protein L (ACTIGEN) wasimmobilized onto a Sensor chip CM4 (GE Healthcare) by an amine couplingmethod, and antibodies of interest were captured onto the chip. Then, adiluted mouse FcRn solution and a running buffer as a blank wereinjected, and mouse FcRn was allowed to interact with the antibodiescaptured onto the sensor chip. The running buffer used was 50 mmol/lsodium phosphate, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.0. Thebuffer was also used to dilute mouse FcRn. 10 mmol/l glycine-HCl (pH1.5) was used for regeneration. All measurements were carried out at 25°C. The binding rate constant ka (1/Ms) and dissociation rate constant kd(1/s), which are kinetic parameters, were calculated from thesensorgrams obtained by the measurement, and the KD (M) of each antibodyfor mouse FcRn was calculated based on the values. Each parameter wascalculated using Biacore T100 or T200 Evaluation Software (GEHealthcare).

The result is shown in Table 14 (KD of human IgG1 or F29 for mouseFcRn). F29 was demonstrated to have increased mouse FcRn-bindingactivity under a neutral pH range condition (pH 7.0).

TABLE 14 ANTIBODY mFcRn KD (M) AMINO ACID SUBSTITUTION HUMAN IgG1 ND F298.5E−08 I332V/N434Y

In Vivo Infusion Test Using Normal Mouse

An infusion pump (MODEL2004, alzet MINI-OSMOTIC PUMP), filled withsoluble human IL-6 receptor, was subcutaneously implanted into the backof a normal mouse (C57BL/6J mouse, Charles River Japan) to create ananimal model with plasma concentration of soluble human IL-6 receptormaintained in the steady state. In the animal model, the anti-human IL-6receptor antibody was administered to assess the in vivo kinetics ofsoluble human IL-6 receptor after antibody administration. To suppressthe production of neutralizing antibodies against soluble human IL-6receptor, an anti-mouse CD4 monoclonal antibody (in-house preparation)was administered once at 20 mg/kg into the tail vein. Then, an infusionpump containing 92.8 μg/ml soluble human IL-6 receptor was implantedsubcutaneously on the back of mice. Three days after implantation of theinfusion pump, 6RKE02-IgG1 and 6RKE02-F29 were administered once at 1mg/kg subcutaneously on the back of the normal mice. Blood was collectedat appropriate time points after administration of the anti-human IL-6receptor antibody. The blood samples obtained were immediatelycentrifuged at 15,000 rpm for 15 minutes at 4° C. to separate plasma.The separated plasma was stored in a freezer set to −20° C. or loweruntil the time of measurement.

hsIL-6R concentration in mouse plasma was determined usingelectrochemiluminescence method. An hsIL-6R calibration curve sampleprepared at 2,000, 1,000, 500, 250, 125, 62.5, or 31.25 pg/mL, and amouse plasma measurement sample diluted by 50-fold or above, were mixedwith a monoclonal anti-human IL-6R antibody (R&D) ruthenated withSULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-humanIL-6R antibody (R&D), and tocilizumab, followed by overnight reaction at37° C. Tocilizumab was prepared at a final concentration of 333 μg/mL.Subsequently, the reaction solution was dispensed into an MA400 PRStreptavidin Plate (Meso Scale Discovery). In addition, after washingoff the reaction solution that was allowed to react at room temperaturefor 1 hour, Read Buffer T (×4) (Meso Scale Discovery) was dispensed.Subsequently, the reaction solution was immediately subjected tomeasurement using a SECTOR PR 400 Reader (Meso Scale Discovery). Theconcentration of hsIL-6R was calculated from the response of thecalibration curve using the SOFTmax PRO analysis software (MolecularDevices).

The time course of the monitored human IL-6 receptor concentration isshown in FIG. 12. The plasma hsIL-6R concentration was not reduced inthe antibody non-administered group and the 6RKE02-IgG1 administeredgroup. By contrast, the plasma hsIL-6R concentration was found to bemarkedly reduced in the group administered with 6RKE02-F29 that binds tothe IL-6 receptor in a pH-dependent manner and has the activity ofbinding mouse FcRn in a neutral range.

In Vivo Drug Efficacy Test Using Normal Mice (Test 1)

Whether 6RKE02-F29 that has no neutralizing activity in vitro canachieve the in vivo pharmaceutical effect by eliminating hsIL-6R fromthe plasma was assessed by using a normal mice model (C57BL/6J Jclmice). It is known that, when a mixed solution of hIL-6 and hsIL-6R isadministered to mice, the following two kinds of signaling aretriggered: trans-signaling by the binding of hIL-6/hsIL-6R complexes tomouse gp130; and classical-signaling by the binding of hIL-6 to mousemembrane IL-6R followed by binding to mouse gp130; thus, the productionof serum amyloid A (SAA) is induced, resulting in an increase of theplasma SAA concentration.

6RKE02-F29 was intravenously administered at 0, 1, 10, and 30 mg/kg toC57BL/6J Jcl mice (female), and, one hour after administration, hIL-6and hsIL-6R were intravenously administered at 4 μg/kg and 7 μg/kg,respectively, as a mixture. Six hours after the second intravenousadministration, blood was collected to determine the plasma SAAconcentration by ELISA. To rule out the effect of endogenous mouse IL-6Ron the plasma SAA concentration, MR16-1 (rat anti-mouse IL-6R antibody)was intravenously administered at 20 mg/kg to all mice simultaneouslywith the test substance. Since the trans-signaling alone is induced byadministering hIL-6/hsIL-6R, it is possible to assess whether the invivo pharmaceutical effect can be achieved through eliminating hsIL-6Rfrom the plasma. Exclusion of the effect of endogenous mouse IL-6R wasconfirmed by intravenously administering hIL-6 at 4 μg/kg to thevehicle-administered group that was intravenously administered withMR16-1 at 20 mg/kg.

The plasma SAA concentration was measured using the SAA Mouse ELISA Kit(catalog NO. KMA0021, Life Technologies Corporation) according to theprotocol attached to the kit. The plasma SAA concentration six hoursafter antibody administration is shown in FIG. 13. Meanwhile, the plasmahsIL-6R concentration six hours after antibody administration is shownin Table 15.

TABLE 15 samples ng/mL 6RKE02-F29 (F29) 30 mg/kg N.D. 6RKE02-F29 (F29)10 mg/kg 0.38 6RKE02-F29 (F29) 1 mg/kg 3.22 IL6/IL6R 17.35  IL6 N.D.

It was confirmed that 6RKE02-F29 reduced the plasma hsIL-6Rconcentration in a dose-dependent manner and thus had the effect toreduce the plasma SAA concentration.

In Vivo Drug Efficacy Test Using Normal Mice (Test 2)

6RKE02-F29 was intravenously administered at 0, 10, and 30 mg/kg toC57BL/6J Jcl mice (female). In test 2, 24 hours after administration,hIL-6 and hsIL-6R were intravenously administered at 4 μg/kg and 7μg/kg, respectively, as a mixture. Six hours after the secondintravenous administration, the blood was collected to determine theplasma SAA concentration by ELISA. To rule out the effect of endogenousmouse IL-6R on the plasma SAA concentration, MR16-1 (rat anti-mouseIL-6R antibody) was intravenously administered at 20 mg/kg to all micesimultaneously with the test substance. Exclusion of the effect ofendogenous mouse IL-6R was confirmed by intravenously administeringhIL-6 at 4 μg/kg to the vehicle-administered group that wasintravenously administered with MR16-1 at 20 mg/kg.

The plasma SAA concentration six hours after antibody administration isshown in FIG. 14. Meanwhile, the plasma hsIL-6R concentration six hoursafter antibody administration is shown in Table 16.

TABLE 16 samples ng/mL 6RKE02-F29 (F29) 30 mg/kg 0.22 6RKE02-F29 (F29)10 mg/kg 0.82 IL6/IL6R 27.50 IL6 N.D.

It was confirmed that 6RKE02-F29 reduced the plasma hsIL-6Rconcentration in a dose-dependent manner and thus had the effect toreduce the plasma SAA concentration.

Tests 1 and 2 revealed that, although 6RKE02-F29 does not have in vitroinhibitory activity against the hsIL-6R-mediated trans-signaling, it canexhibit the in vivo inhibitory effect against the trans-signaling byeliminating hsIL-6R from the plasma.

Based on this finding, it can be said that, when using an antibody thatbinds to a target antigen in a pH-dependent manner and which hasFcRn-binding activity in a neutral pH range, even if the antibody doesnot have the neutralizing activity to a specific epitope in vitro, thein vivo pharmaceutical effect (inhibitory effect) can be exhibited byeliminating the target antigen from the plasma. Ordinary antigen-bindingmolecules including monoclonal antibodies can only bind to a singleepitope. Meanwhile, in certain antigens, there are several antigenicsites to be neutralized; ordinary monoclonal antibodies can neutralizethe action of a single epitope, but cannot neutralize the action ofother epitopes of such antigens. Even in this case, as shown in thisExample, the action of all epitopes can be substantially inhibited wheneliminating the antigen from the plasma by using a monoclonal antibodythat binds to the target antigen in a pH-dependent manner and which hasFcRn-binding activity in a neural pH range.

Reference Example 6 Preparation of Antigen-Binding Molecules Whose MouseFcγR-Binding Activity Under a Neutral pH Range Condition is Higher thanthe Binding Activity of Native Human IgG Fc Region

(6-1) pH-Dependent Human IL-6 Receptor-Binding Antibodies

H54/L28-IgG1 which comprises H54-IgG1 (SEQ ID NO: 113) and L28-CK (SEQID NO: 114) described in WO2009/125825 is a humanized anti-IL-6 receptorantibody. Meanwhile, Fv4-IgG1 which comprises VH3-IgG1 (SEQ ID NO: 4)and VL3-CK (SEQ ID NO: 122) is a humanized anti-IL-6 receptor antibodyresulting from conferring, to H54/L28-IgG1, the property of binding tosoluble human IL-6 receptor in a pH-dependent manner (which binds at pH7.4 and dissociates at pH 5.8). The in vivo mouse test described inWO2009/125825 demonstrated that, in the group administered with amixture of Fv4-IgG1 and soluble human IL-6 receptor as the antigen, theelimination of soluble human IL-6 receptor from plasma was significantlyaccelerated as compared to the group administered with a mixture ofH54/L28-IgG1 and soluble human IL-6 receptor as the antigen.

The soluble human IL-6 receptor bound to H54/L28-IgG1 is, together withthe antibody, recycled to plasma by FcRn. Meanwhile, Fv4-IgG1dissociates soluble human IL-6 receptor under the acidic condition inthe endosome, and the dissociated soluble human IL-6 receptor isdegraded in the lysosomes, thus this enables considerable accelerationof the elimination of soluble human IL-6 receptor. After binding to FcRnin the endosome, Fv4-IgG1 is recycled to the plasma. Since the recycledantibody can bind to soluble human IL-6 receptor again, the antibodyrepeatedly binds to the antigen (soluble human IL-6 receptor) and isrecycled by FcRn to the plasma. It is thought that, as a result, asingle antibody molecule can bind repeatedly several times to solublehuman IL-6 receptor (FIG. 15).

(6-2) Preparation of an Anti-Human IL-6 Receptor Antibody with EnhancedMouse FcγR Binding and Anti-Human IL-6 Receptor Antibody without MouseFcγR Binding

VH3-IgG1-F1022 (SEQ ID NO: 124), an antigen-binding molecule withenhanced mouse FcγR binding, was prepared by substituting Asp for Lys atposition 326 (EU numbering) and Tyr for Leu at position 328 (EUnumbering) in VH3-IgG1. Fv4-IgG1-F1022 containing VH3-IgG1-F1022 as theheavy chain and VL3-CK as the light chain was produced using the methoddescribed in Reference Example 1.

Meanwhile, VH3-IgG1-F760 (SEQ ID NO: 123), an antigen-binding moleculewithout mouse FcγR binding, was prepared by substituting Arg for Leu atposition 235 and Lys for Ser at position 239 (EU numbering) in VH3-IgG1.Fv4-IgG1-F760 containing VH3-IgG1-F760 as the heavy chain and VL3-CK asthe light chain was produced using the method described in ReferenceExample 1.

(6-3) Assessment of Mouse FcγR-Binding Activity

VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F1022, and VH3/L(WT)-IgG1-F760, whichcontain VH3-IgG1, VH3-IgG1-F1022, and VH3-IgG1-F760 as the heavy chain,respectively, and L(WT)-CK (SEQ ID NO: 5) as the light chain, wereproduced using the method described in Reference Example 1. Theseantibodies were kinetically analyzed for their mouse FcγR binding asdescribed below.

(6-4) Kinetic Analysis of Mouse FcγR Binding

The binding of antibodies to mouse FcγRI, FcγRIIb, FcγRIII, and FcγRIV(hereinafter, referred to as mouse FcγRs) (R&D systems, Sino Biological,or prepared by the method described in Reference Example 2) waskinetically analyzed using Biacore T100 and T200 (GE Healthcare). Anappropriate amount of protein L (ACTIGEN or BioVision) was immobilizedonto a Sensor chip CM4 (GE Healthcare) by an amine coupling method, andantibodies of interest were captured thereto. Then, diluted solutions ofmouse FcγRs and a running buffer as a blank were injected, and the mouseFcγRs were allowed to interact with antibodies captured onto the sensorchip. The running buffer used was 20 mmol/l ACES, 150 mmol/l NaCl, 0.05%(w/v) Tween20, pH 7.4. This buffer was also used to dilute the mouseFcγRs. The sensor chip was regenerated using 10 mmol/l glycine-HCl, pH1.5. All measurements were carried out at 25° C. The binding rateconstant ka (1/Ms) and dissociation rate constant kd (1/s), which arekinetic parameters, were calculated from the sensorgrams obtained by themeasurement. KD (M) of each antibody for human FcγR was calculated basedon the values. Each parameter was calculated using Biacore T100 or T200Evaluation Software (GE Healthcare).

The result shown in Table 17 was obtained by the measurement. VH3/L(WT)-IgG1-F1022 was demonstrated to have increased binding activity tomFcγRI, mFcγRIIb, and mFcγRIII as compared to VH3/L (WT)-IgG1. RegardingVH3/L (WT)-IgG1-F760, the binding to the various mouse FcγRs wasundetectable, demonstrating that VH3/L (WT)-IgG1-F760 lacks the bindingactivity to the various mouse FcγRs. In the table, VH3/L (WT)-IgG1 isabbreviated as IgG1; VH3/L (WT)-IgG1-F1022 is abbreviated as F1022; andVH3/L (WT)-IgG1-F760 is abbreviated as F760.

TABLE 17 VARIANT KD (M) NAME mFcγRI mFcγRII mFcγRIIII mFcγRIV IgG15.3E−08 9.8E−07 2.4E−06 8.6E−08 F1022 7.6E−09 1.0E−08 5.5E−09 1.4E−07F760 NOT NOT NOT NOT DETECTED DETECTED DETECTED DETECTED(6-5) Preparation of Antibodies with Low Fucose Content

Known methods for increasing the FcγR-binding activity of antibodiesinclude methods for making sugar chains linked to an antibody be sugarchains with low fucose content (J. Biol. Chem. (2003) 278, 3466-3473) inaddition to methods for introducing an amino acid alteration into the Fcregion of an antibody. An Fv4-IgG1 with low fucose content (hereinafter,abbreviated as Fv4-IgG1-Fuc) was produced by expressing Fv4-IgG1 usingfucose transporter gene-deficient CHO cells (WO2006/067913) as hostcells according to the method described in Reference Example 1. It hasbeen reported that, of the mFcγRs (mouse Fcγ receptors), antibodies withlow fucose content have selectively increased FcγRIV-binding activity(Science, 2005, 310 (5753) 1510-1512).

Reference Example 7 Effect of Eliminating Antigens from Plasma byAntigen-Binding Molecules Whose FcγR-Binding Activity is Higher than theBinding Activity of Native Human IgG Fc Region

(7-1) Effect of H54/L28-IgG1 and Fv4-IgG1 to Eliminate Antigens fromPlasma

H54/L28-IgG1, which is an anti-human IL-6 receptor antibody, andFv4-IgG1 having the property of binding to human IL-6 receptor in apH-dependent manner were produced by the method described in ReferenceExample 1. In vivo infusion tests were carried out using the producedH54/L28-IgG1 and Fv4-IgG1 by the method described below.

(7-1-1) In Vivo Infusion Tests Using Human FcRn Transgenic Mice

An animal model in which the soluble human IL-6 receptor concentrationis maintained constant in plasma was created by implanting an infusionpump (MINI-OSMOTIC PUMP MODEL2004, alzet) containing soluble human IL-6receptor under the skin on the back of human FcRn transgenic mice(B6.mFcRn−/−.hFcRn Tg line 32+/+mouse, Jackson Laboratories, MethodsMol. Biol. (2010) 602, 93-104). The in vivo dynamics afteradministration of an anti-human IL-6 receptor antibody was assessed inthe animal model. To suppress the production of neutralizing antibodiesagainst soluble human IL-6 receptor, an anti-mouse CD4 monoclonalantibody (prepared by a known method) was administered once at 20 mg/kginto the tail vein. Then, an infusion pump containing 92.8 μg/ml solublehuman IL-6 receptor was subcutaneously implanted on the back of themice. Three days after implantation of the infusion pump, an anti-humanIL-6 receptor antibody was administered once at 1 mg/kg into the tailvein. The blood was collected from the mice 15 minutes, seven hours, oneday, two days, four days, and seven days after administration of theanti-human IL-6 receptor antibody. Immediately, the collected blood wascentrifuged at 15,000 rpm and 4° C. for 15 minutes to prepare plasma.The isolated plasma was stored in a freezer set at −20° C. or belowuntil use.

(7-1-2) Determination of the Soluble Human IL-6 Receptor (hsIL-6R)Concentration in Plasma by an Electrochemiluminescent Method

The hsIL-6R concentrations in mouse plasma were determined by anelectrochemiluminescent method. hsIL-6R standard curve samples preparedat 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg/ml and assay samples ofmouse plasma diluted 50 times or more were mixed with MonoclonalAnti-human IL-6R Antibody (R&D) which had been ruthenated with SULFO-TAGNHS Ester (Meso Scale Discovery), Biotinylated Anti-human IL-6 RAntibody (R&D), and Tocilizumab. The mixtures were incubated at 37° C.overnight. Tocilizumab was prepared at a final concentration of 333μg/ml. Then, the reaction mixtures were aliquoted in an MA400 PRStreptavidin Plate (Meso Scale Discovery). The solution reacted at roomtemperature for one hour was washed out, and then Read Buffer T (×4)(Meso Scale Discovery) was aliquoted. Immediately thereafter, themeasurement was carried out using SECTOR PR 400 Reader (Meso ScaleDiscovery). The concentration of hsIL-6R was determined based on theresponse of the standard curve using analysis software SOFTmax PRO(Molecular Devices).

A time course of the monitored human IL-6 receptor concentration isshown in FIG. 16. As compared to H54/L28-IgG1, Fv4-IgG1 that binds tohuman IL-6 receptor in a pH-dependent manner could reduce the human IL-6receptor concentration, but could not reduce it below the baselinewithout antibody administration. That is, the administered antibodywhich binds to an antigen in a pH-dependent manner could not reduce theantigen concentration in plasma below the level prior to antibodyadministration.

(7-2) the Effect of Eliminating an Antigen from Plasma by an Antibodywith Increased or Reduced FcγR-Binding Activity

Whether the time course of human IL-6 receptor concentration isinfluenced by increasing or reducing the FcγR-binding activity ofFv4-IgG1, which is a pH-dependent human IL-6 receptor-binding antibody,was assessed by the method described below. Using Fv4-IgG1,Fv4-IgG1-F760, Fv4-IgG1-F1022, and Fv4-IgG1-Fuc prepared as described inReference Example 6, in vivo infusion tests were performed by the methoddescribed below.

(7-2-1) In Vivo Infusion Tests Using Human FcRn Transgenic Mice

A animal model in which the soluble human IL-6 receptor concentration ismaintained constant in plasma was created by implanting an infusion pump(MINI-OSMOTIC PUMP MODEL2004, alzet) containing soluble human IL-6receptor under the skin on the back of human FcRn transgenic mice(B6.mFcRn−/−.hFcRn Tg line32+/+mouse, Jackson Laboratories, Methods MolBiol. (2010) 602, 93-104). In the animal model, an anti-human IL-6receptor antibody was administered simultaneously with Sanglopor (CSLBehring) which is a human immunoglobulin preparation, to assess the invivo dynamics of the soluble human IL-6 receptor after antibodyadministration. To suppress the production of neutralizing antibodiesagainst soluble human IL-6 receptor, an anti-mouse CD4 monoclonalantibody (prepared by a known method) was administered once at 20 mg/kginto the tail vein. Then, an infusion pump containing 92.8 μg/ml solublehuman IL-6 receptor was subcutaneously implanted on the back of themice. Three days after implantation of the infusion pump, an anti-humanIL-6 receptor antibody and Sanglopor were administered once at 1 mg/kgand 1000 mg/kg, respectively, into the tail vein. The blood wascollected from the mice 15 minutes, seven hours, one day, two days, fourdays, seven days, 14 days, and 21 days after administration of theanti-human IL-6 receptor antibody. The blood was collected from the mice15 minutes, seven hours, one day, two days, three days, seven days, 14days, and 21 days after administration of the anti-human IL-6 receptorantibody. Immediately, the collected blood was centrifuged at 15,000 rpmand 4° C. for 15 minutes to prepare the plasma. The isolated plasma wasstored in a freezer set at −20° C. or below until use.

(7-2-2) Determination of the Soluble Human IL-6 Receptor (hsIL-6R)Concentration in Plasma by an Electrochemiluminescent Method

The hsIL-6R concentrations in mouse plasma were determined by the sameelectrochemiluminescent method as described in (7-1-2).

The result is shown in FIG. 17. The time course of human IL-6 receptorconcentration in plasma of mice administered with Fv4-IgG1-F760, fromwhich the mouse FcγR binding of Fv4-IgG1 is deleted, was demonstrated tobe comparable to that in mice administered with Fv4-IgG1. The cytotoxicactivity to a membrane antigen depends on the FcγR binding, and thus thecytotoxic activity is lost when eliminating the FcγR binding. On theother hand, even when administering an antibody, from which mouse FcγRbinding is deleted, against human IL-6 receptor which is a solubleantigen, there was no effect on the time course of human IL-6 receptorconcentration in the plasma of the administered mice. Thus, it would bethought that the FcγR binding of an antibody against the soluble antigenhas no contribution to the time course of antigen concentration in theplasma of mice administered with the antibody.

Surprisingly, however, the human IL-6 receptor concentration in theplasma of mice administered with Fv4-IgG1-F1022 with enhanced mouse FcγRbinding was considerably reduced as compared to the human IL-6 receptorconcentration in the plasma of mice administered with Fv4-IgG1. As tothe degree of reduction, the concentration was confirmed to be decreasedbelow the base-line human IL-6 receptor concentration without antibodyadministration. In particular, the human IL-6 receptor concentration inthe plasma of mice administered with Fv4-IgG1-F1022 was reduced down toabout 1/100 three days after administration as compared to the case ofFv4-IgG1 administration. This finding demonstrates that, byadministering to mice an antibody that binds to human IL-6 receptor in apH-dependent manner and whose FcγR binding has been enhanced, the humanIL-6 receptor concentration in the plasma of the mice can besignificantly reduced, and as to the degree of reduction, the antigenconcentration in plasma can be reduced below the level before antibodyadministration.

Furthermore, it was also demonstrated that, as compared to miceadministered with Fv4-IgG1, the human IL-6 receptor concentration inplasma was reduced in mice administered with Fv4-IgG1-Fuc which hassugar chains with low fucose content and with increased mouse FcγRIV-binding activity. In particular, the human IL-6 receptorconcentration in the plasma of mice administered with Fv4-IgG1-Fuc wasreduced down to about ½ seven days after administration as compared tothe case of Fv4-IgG1 administration. The above finding demonstratesthat, by administering to mice a pH-dependent antigen-binding moleculethat binds to human IL-6 receptor in a pH-dependent manner and whoseFcγR binding has been enhanced, the soluble antigen concentration in theplasma of the mice can be reduced. Methods for enhancing the FcγRbinding are not particularly limited to introduction of amino acidalterations. It was demonstrated that such enhancement can be achieved,for example, by using a human IgG Fc region to which a sugar chain withlow fucose content is linked at position 297 (EU numbering); however,the effect of Fv4-IgG1-Fuc to reduce antigen concentration was smallerthan Fv4-F1022. Thus, it would be thought that, of several FcγRs (FcγRI,II, III, and IV for mouse), mFcγIV, to which the binding of Fv4-IgG1-Fucis enhanced, does not have a large contribution to the reduction ofantigen concentration as an FcγR.

Thus, it was revealed that, by administering to an individual anantibody that binds to a soluble antigen in a pH-dependent manner andwhose FcγR binding has been enhanced, the soluble antigen concentrationin the plasma of the individual can be markedly reduced.

Without being bound by a particular theory, the unexpected reduction ofsoluble antigen concentration in plasma, which was observed whenadministering an antigen-binding molecule whose FcγR binding has beenenhanced and that comprises an antigen-binding domain whoseantigen-binding activity is altered depending on the ion concentrationcondition such as pH and an FcRn-binding domain that has FcRn-bindingactivity under an acidic pH range condition, can be explained asfollows.

IgG antibodies that are non-specifically incorporated into cells returnto the cell surface by binding to FcRn under the acidic condition in theendosome, and then dissociate from FcRn under the neutral condition inplasma. In such a case, when an antibody that neutralizes the functionof a soluble antigen by binding to the antigen is administered to micein which the concentration of the soluble antigen is maintained constantin plasma, the soluble antigen in plasma forms a complex with theantibody. The soluble antigen incorporated into cells while remaining asthe complex is thought to be recycled, in a state bound to the antibody,to the plasma together with the antibody, because the Fc region of theantibody binds to FcRn under the acidic condition in the endosome.

Meanwhile, when the antibody against the soluble antigen is an antibodythat binds to the antigen in a pH-dependent manner (i.e., an antibodythat dissociates the soluble antigen under the acidic condition in theendosome), the soluble antigen that is non-specifically incorporatedinto cells while remaining as a complex with the antibody, isdissociated from the antibody in the endosome and degraded in thelysosome; thus, the soluble antigen is not recycled to the plasma. Thatis, it is thought that Fv4-IgG1 incorporated as a complex with thesoluble antigen into cells can dissociate the soluble antigen in theendosome and thus accelerate the elimination of the soluble antigen.

As described above, antigen-binding molecules such as Fv4-IgG1, whichcontain an antigen-binding domain whose antigen-binding activity isaltered depending on the ion concentration, are thought to be capable ofbinding to antigens repeatedly several times. The effect to acceleratethe elimination of soluble antigens from the plasma by dissociating themin the endosome is thought to depend on the rate of incorporation of theantigen/antigen-binding molecule complex into the endosome. Anantigen-binding molecule whose binding activity to various FcγRs hasbeen increased and that contains an antigen-binding domain whoseantigen-binding activity is altered depending on the condition of ionconcentration, is actively incorporated into cells by binding to variousFcγRs expressed on the cell membrane, and can be shuttled back to plasmaby recycling via the binding between FcRn and the FcRn-binding domaincomprised in the molecule, which has FcRn-binding activity under anacidic pH range condition. That is, it is thought that, since the aboveantigen-binding molecule which forms a complex with a soluble antigen inplasma is actively incorporated into cells via FcγR expressed on thecell membrane, its effect to accelerate the elimination of the solubleantigen from plasma is more markedly shown than antigen-bindingmolecules whose binding activity to various FcγRs has not beenincreased.

The FcγR-binding activity of an antibody that binds to a membraneantigen plays an important role in the cytotoxic activity of theantibody. Thus, when it is necessary for an antibody used as apharmaceutical agent to have cytotoxic activity, a human IgG1 isotypewith strong FcγR-binding activity is used. In addition, techniques toenhance the cytotoxic activity of such antibodies by increasing theFcγR-binding activity of the antibodies are used commonly in the art.

Meanwhile, the role of the FcγR-binding activity of antibodies that bindto soluble antigens and which are used as pharmaceutical agents has notbeen known in the art. There has been no sufficient assessment on whatdifference in the effect on the living organism administered with theantibodies is caused by the difference in the FcγR-binding activitybetween human IgG1 with high FcγR-binding activity and human IgG2 andhuman IgG4 with low FcγR-binding activity. Actually, it was demonstratedin the present Example that there was no influence on the time course ofsoluble antigen concentration in the plasma of the individualsadministered with an antibody that lacks FcγR-binding activity.Meanwhile, in the present invention, it was revealed that the solubleantigen concentration was significantly reduced in the plasma of theindividuals administered with an antigen-binding molecule whoseFcγR-binding activity has been increased and which contains anantigen-binding domain whose soluble antigen-binding activity is altereddepending on the ion concentration condition. Specifically, it can besaid that the present inventors revealed for the first time the benefitof the enhancement of FcγR binding by combining an FcRn-binding domainthat has FcRn-binding activity under an acidic pH range condition withan antigen-binding domain whose soluble antigen binding is altereddepending on the ion concentration condition, comprised in anantigen-binding molecule targeted to a soluble antigen.

Reference Example 8 Effect of Eliminating Antigens from Plasma byAntigen-Binding Molecules Whose FcγR-Binding Activity is Greater thanthat of Native Human IgG Fc Region and Whose Human FcRn-Binding Activityhas been Increased Under an Acidic pH Range Condition

(8-1) Preparation of Antigen-Binding Molecules Whose FcγR-BindingActivity is Greater than the Binding Activity of Native Human IgG FcRegion and Whose Human FcRn-Binding Activity has been Increased Under anAcidic pH Range Condition

A reported method for improving the retention of IgG antibody in plasmais to improve the FcRn binding under an acidic pH range condition. It isthought that, when the FcRn binding under an acidic pH range conditionis improved by introducing an amino acid substitution into the Fc regionof an IgG antibody, this increases the recycling efficiency from theendosome to plasma, resulting in an improvement of the plasma retentionof the IgG antibody.

There are many reports on amino acid alterations to improve the plasmaretention by improving the human FcRn-binding activity under an acidicpH range condition. Such alterations include, for example:

the method for substituting Leu for Met at position 428 and Ser for Asnat position 434 (EU numbering) in an IgG antibody (Nat. Biotechnol,(2010) 28, 157-159); the method for substituting Ala for Asn at position434 (Drug. Metab. Dispos. (2010) 38 (4), 600-605); the method forsubstituting Tyr for Met at position 252, Thr for Ser at position 254,and Glu for Thr at position 256 (J. Biol. Chem. (2006) 281,23514-23524); the method for substituting Gln for Thr at position 250and Leu for Met at position 428 (J. Immunol. (2006) 176 (1) 346-356);the method for substituting His for Asn at position 434 (Clin. Pharm. &Ther. (2011) 89 (2) 283-290.); and WO2010/106180; WO2010/045193;WO2009/058492; WO2008/022152; WO2006/050166, WO2006/053301,WO2006/031370; WO2005/123780; WO2005/047327; WO2005/037867;WO2004/035752; and WO2002/060919.

VH3-IgG1-F1093 (SEQ ID NO: 125) with a substitution of Leu for Met atposition 428 and Ser for Asn at position 434 (EU numbering) inVH3-IgG1-F1022 was prepared to improve the pharmacokinetics ofFv4-IgG1-F1022 that was demonstrated to produce, when administered, theeffect of significantly reducing the soluble antigen concentration inplasma, as described in Reference Example 7. Fv4-IgG1-F1093 comprisingVH3-IgG1-F1093 as the heavy chain and VL3-CK as the light chain wasconstructed using the method described in Reference Example 1.

(8-2) Effect of Eliminating Antigens from Plasma by Antigen-BindingMolecules Whose FcγR-Binding Activity is Greater than that of NativeHuman IgG Fc Region and Whose Human FcRn-Binding Activity has beenIncreased Under an Acidic pH Range Condition

An in vivo infusion test was carried out for Fv4-IgG1-F1093 by the samemethod as described in (7-1-1) using human FcRn transgenic mice in whichthe soluble human IL-6 receptor concentration is maintained constant inplasma. Soluble human IL-6 receptor concentrations in the plasma of themice were determined by the method described in (7-1-2). The result isshown in FIG. 18.

(8-2-1) Determination of the Anti-Human IL-6 Receptor AntibodyConcentration in Plasma by ELISA

Anti-human IL-6 receptor antibody concentrations in the plasma of themice were determined by ELISA. First, an anti-Fv4 ideotype antibody(in-house preparation) was aliquoted in a Nunc-Immuno Plate, MaxiSorp(Nalge nunc International). The plate was allowed to stand at 4° C.overnight to prepare a plate immobilized with the anti-Fv4 ideotypeantibody. Standard curve samples with a concentration of 6.4, 3.2, 1.6,0.8, 0.4, 0.2, or 0.1 μg/ml plasma, and assay samples of mouse plasmadiluted 100 times or more were prepared. 100 μl of the standard curveand plasma assay samples were mixed with 200 μl of 20 ng/ml hsIL-6R and2 mg/ml Sanglopor. This was allowed to stand at one hour at roomtemperature, and then aliquoted into a plate immobilized with ananti-Fv4 ideotype antibody. The plate was allowed to stand at roomtemperature for one hour. Then, Biotinylated Anti-human IL-6 R Antibody(R&D) was reacted at room temperature for one hour. Next,Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) wasreacted at room temperature for one hour. Chromogenic reaction wasperformed using TMB One Component HRP Microwell Substrate (BioFXLaboratories) as a substrate. After terminating the reaction with 1Nsulfuric acid (Showa Chemical), the absorbance at 450 nm was measuredwith a microplate reader. The concentrations in mouse plasma weredetermined based on the absorbance of the standard curve using theanalysis software SOFTmax PRO (Molecular Devices). The result is shownin FIG. 19.

(8-3) Improvement of Pharmacokinetics by Increasing the HumanFcRn-Binding Activity Under an Acidic pH Range Condition

As shown in FIG. 19, in the group administered with Fv4-IgG1-F1022resulting from the enhancement of the FcγR-binding activity of Fv4-IgG1under a neutral pH range condition, the plasma retention of theadministered antibody was demonstrated to be reduced as compared to thegroup administered with Fv4-IgG1. Meanwhile, in the group administeredwith Fv4-IgG1-F1093 resulting from the enhancement of the humanFcRn-binding activity of Fv4-IgG1-F1022 under an acidic pH rangecondition, the plasma retention of the administered antibody wasdemonstrated to be significantly improved as compared to the groupadministered with Fv4-IgG1-F1022.

Furthermore, as shown in FIG. 18, the time course of the soluble humanIL-6 receptor concentration in the plasma of theFv4-IgG1-F1022-administered group was equivalent to that of theFv4-IgG1-F1093-administered group, up to three days after antibodyadministration. On day three after administration, as compared to theFv4-IgG1-administered group, the soluble human IL-6 receptorconcentration in plasma was reduced as much as 100 times in both of theFv4-IgG1-F1022 and Fv4-IgG1-F1093-administered groups. However, on dayseven after antibody administration, the soluble human IL-6 receptorconcentration in plasma was observed to be elevated in theFv4-IgG1-F1022-administered group as compared to on day three afteradministration. On the other hand, in the Fv4-IgG1-F1093-administeredgroup, an increase in the plasma concentration of soluble human IL-6receptor was not observed, showing that the effect to reduce the solublehuman IL-6 receptor concentration was sustained in this administrationgroup.

Specifically, Fv4-IgG1-F1093, when administered, reduced the solublehuman IL-6 receptor concentration in the plasma of the administeredindividual down to about 1/100 as compared to Fv4-IgG1, and in addition,it sustained this condition for a long period. Thus, Fv4-IgG1-F1093 wasdemonstrated to be a highly excellent antigen-binding molecule. Withoutbeing bound by a particular theory, the phenomenon observed herein canbe explained as follows. Fv4-IgG1-F1022 in which the FcγR-bindingactivity of Fv4-IgG1 has been increased under a neutral pH rangecondition is thought to be incorporated in a large amount mainly intocells expressing FcγR on the cell membrane. The incorporated antibody istransferred into the endosome, and by binding to FcRn in the endosome,the antibody is recycled to the plasma. When the FcRn-binding activityof the antibody is not high enough under the condition at acidic pH inthe endosome, the antibody incorporated into the endosome is thought tobe incapable of sufficient recycling. Specifically, a possible reasonfor the reduced plasma retention of Fv4-IgG1-F1022 relative to Fv4-IgG1would be that the FcRn-binding activity under an acidic pH rangecondition is insufficient for sufficient recycling of theendosome-incorporated antibody to the plasma by FcRn binding, and theantibody that was not recycled was degraded in the lysosome.

On the other hand, as with Fv4-IgG1-F1022, Fv4-IgG1-F1093 resulting fromthe enhancement of the human FcRn-binding activity of Fv4-IgG1-F1022under an acidic pH range condition is thought to be incorporated in alarge amount mainly into cells expressing FcγR on the cell membrane. Anantibody incorporated and transferred into the endosome is recycled tothe plasma by binding to FcRn in the endosome. Since its humanFcRn-binding activity under an acidic pH range condition is enhanced,Fv4-IgG1-F1093 is thought to have sufficient FcRn-binding activity inthe endosome. Thus, after incorporation into cells, most ofFv4-IgG1-F1093 is recycled to the plasma. Thus, it would be thought thatthe plasma retention of Fv4-IgG1-F1093 was improved in administeredindividuals as compared to Fv4-IgG1-F1022.

On the other hand, it has been known that the plasma retention ofordinary antibodies is improved when their FcRn-binding activity isimproved under an acidic pH range condition. However, it is thoughtthat, when the antibody retention in plasma is improved, the plasmaretention of antibody-bound antigens is also improved, and this resultsin an increase of the antigen concentration in plasma. In actual, asdescribed in WO2010/088444, Antibody 18E introduced with the alterationYTE into Antibody 18, which is a human IgG1 antibody against IL-6, toincrease the FcRn-binding activity under an acidic pH range condition,showed improved antibody retention in the plasma of cynomolgus monkeys,and at the same time, the concentration of the IL-6 antigen was alsoelevated in the plasma.

Surprisingly, however, when administering Fv4-IgG1-F1093 introduced withan alteration similar to YTE for increasing the FcRn-binding activityunder an acidic pH range condition into Fv4-IgG1-F1022 that binds to theantigen in a pH-dependent manner and has increased FcγR-bindingactivity, the plasma retention of the antibody was significantlyimproved in the administered individuals without increasing theconcentration of soluble human IL-6 receptor which is the antigen.Rather, on day seven after antibody administration, the soluble humanIL-6 receptor concentration remained low in the individuals administeredwith Fv4-IgG1-F1093 as compared to those administered withFv4-IgG1-F1022.

Without being bound by a particular theory, the phenomenon observedherein can be explained as follows. When administered to a livingorganism, an antibody without pH-dependent antigen binding isnon-specifically incorporated into cells. Antigens that remain to bebound to the antibody are recycled to the plasma in the same extent asthe antibody. Meanwhile, for an antibody with increased FcRn-bindingactivity under an acidic pH range condition, the extent of recycling tothe plasma in a living organism administered with the antibody is higherthan that of an antibody without increased FcRn-binding activity, andthis results in an increased extent of recycling of antigens bound tothe antigen to the plasma in the living organism. Thus, due to theimproved plasma retention of the antibody administered in the livingorganism, the plasma concentration of the antigen to which the antibodybinds is thought to be also increased in the living organism.

Meanwhile, when administered to a living organism, an antibody thatbinds to an antigen in a pH-dependent manner and which has increasedFcγR-binding activity is mainly incorporated into cells expressing FcγRon the cell membrane, and this worsens the plasma retention.Furthermore, after being incorporated into the cells while bound to theantibody, the antigen is dissociated from the antibody in the endosomeand then degraded in the lysosome, resulting in a decrease of theantigen concentration in plasma in the living organism. When theFcRn-binding activity is increased under an acidic pH range condition,the antibody retention in plasma, even if worsened due to increasedFcγR-binding activity, is improved by an increase in the rate ofrecycling by FcRn. In this case, since the antigen bound to the antibodythat binds to the antigen in a pH-dependent manner is dissociated fromthe antibody in the endosome and directly degraded in the lysosome, itis not thought that the antigen concentration is increased in theplasma. Furthermore, the improved plasma retention of the antibodyadministered to the living organism is thought to allow the antigenelimination effect of the antibody to be sustained, and the antigenconcentration to be maintained low for a longer period.

The above findings demonstrate that the plasma retention of anadministered antibody is improved in a living organism administered withthe antibody in which the human FcRn-binding activity under an acidic pHrange condition is enhanced in an antigen-binding molecule whoseFcγR-binding activity is higher than that of native human IgG Fc region.Furthermore, it was revealed that, in this case, the antibody retentionin plasma is improved without deteriorating the antigen-eliminationeffect.

Reference Example 9 Further Assessment of the Effect of EliminatingAntigens from Plasma by Antigen-Binding Molecules Whose FcγR-BindingActivity is Greater than that of Native Human IgG Fc Region and WhoseHuman FcRn-Binding Activity has been Increased Under an Acidic pH RangeCondition

(9-1) the Antigen Elimination Effect of an Antibody with IncreasedFcγR-Binding Activity

As described in Reference Example 7, the antigen concentration in plasmawas significantly reduced in the group administered with Fv4-IgG1-F1022with enhanced mouse FcγR binding. Meanwhile, as shown in ReferenceExample 8, the reduced plasma retention observed in theFv4-IgG1-F1022-administered group was markedly improved by increasingthe human FcRn-binding activity of Fv4-IgG1-F1022 under an acidic pHrange condition. Next, the effect of eliminating soluble antigens fromplasma by enhancing mouse FcγR binding and the effect of improving theplasma antibody retention by enhancing the human FcRn binding activityunder an acidic pH range condition were further assessed as describedbelow.

(9-2) Preparation of an Anti-Human IL-6 Receptor Antibody with EnhancedMouse FcγR Binding

VH3-IgG1-F1087 (SEQ ID NO: 145) resulting from substituting Asp for Lysat position 326 (EU numbering) in VH3-IgG1, and VH3-IgG1-F1182 (SEQ IDNO: 148) resulting from substituting Asp for Ser at position 239 and Glufor Ile at position 332 (EU numbering) in VH3-IgG1, were prepared asantigen-binding molecules with enhanced mouse FcγR binding.Fv4-IgG1-F1087 that contains VH3-IgG1-F1087 as the heavy chain andVL3-CK as the light chain, and Fv4-IgG1-F1182 that containsVH3-IgG1-F1182 as the heavy chain and VL3-CK as the light chain, wereproduced using the method described in Reference Example 1.

(9-3) Assessment of Mouse FcγR-Binding Activity

VH3/L (WT)-IgG1-F1087 and VH3/L (WT)-IgG1-F1182 which containVH3-IgG1-F1087 and VH3-IgG1-F1182 as the heavy chain, respectively, andL (WT)-CK (SEQ ID NO: 5) as the light chain, were prepared by the methoddescribed in Reference Example 1. These antibodies, VH3/L(WT)-IgG1-F1022, and VH3/L (WT)-IgG1 were assessed for their mouseFcγR-binding activity by the method described in Reference Example 2.The result is shown in Table 18. In addition, the ratio of the increasein the mouse FcγR-binding activity of each variant relative to the IgG1before alteration is shown in Table 19. In the table, VH3/L (WT)-IgG1 isabbreviated as IgG1; VH3/L (WT)-IgG1-F1022 is abbreviated as F1022;VH3/L (WT)-IgG1-F1087 is abbreviated as F1087; and VH3/L (WT)-IgG1-F1182is abbreviated as F1182.

TABLE 18 VARIANT KD (M) NAME mFcγRI mFcγRIIb mFcγRIII mFcγRIV IgG15.3E−08 9.8E−07 2.4E−06 8.6E−08 F1022 7.6E−09 1.0E−08 5.5E−09 1.4E−07F1087 2.9E−08 5.6E−08 5.2E−08 3.3E−07 F1182 2.4E−09 1.1E−07 4.8E−075.3E−10

TABLE 19 VARIANT RATIO OF BINDING TO IgG1 NAME mFcγRI mFcγRIIb mFcγRIIImFcγRIV IgG1 1.0 1.0 1.0 1.0 F1022 7.0 93.6 440.5 0.6 F1087 1.8 17.546.2 0.3 F1182 22.1 9.1 5.0 162.3

As shown in Table 19, it was demonstrated that F1087 and F1022 hadincreased binding activity to mouse FcγRI, mouse FcγRIIb, and mouseFcγRIII as compared to IgG1, whereas their mouse FcγRIV-binding activitywas not increased. Regarding the binding activity of F1087 to mouseFcγRI, mouse FcγRIIb, mouse FcγRIII, and mouse FcγRIV, the extent of itsincrease was revealed to be smaller than that of F1022. Meanwhile, itwas shown that the binding activity of F1182 to mouse FcγRI and mouseFcγRIV was considerably increased, whereas the extent of increase in itsbinding activity to FcγRIIb and FcγRIII was smaller than those of F1022and F1087. As mentioned above, these three types of variants showedenhanced binding to some mouse FcγRs; however, it was shown that theFcγR to which the binding activity is selectively increased and theextent of the increase vary depending on the variant.

(9-4) the Effect of Eliminating Antigens from Plasma by Fv4-IgG1-F1087and Fv4-IgG1-F1182

By the same method as described in Reference Example 7, in vivo infusiontests using human FcRn transgenic mice were carried out to determine thesoluble human IL-6 receptor concentrations in the plasma of the mice.The result is shown in FIG. 20.

In both of the groups administered with Fv4-IgG1-F1087 andFv4-IgG1-F1182 in vivo, which have increased mouse FcγR-binding activityas compared to Fv4-IgG1, the in vivo plasma concentration of solublehuman IL-6 receptor was able to be reduced as compared to the groupadministered with Fv4-IgG1. The effect to reduce the plasmaconcentration of soluble human IL-6 receptor was high especially in thegroup administered with Fv4-IgG1-F1087 which has enhanced binding tomouse FcγRII and mouse FcγRIII. Meanwhile, the effect of F1182administration to reduce the plasma concentration of soluble human IL-6receptor was small in the group administered with F1182 in vivo whichhas considerably increased binding activity to mouse FcγRI and mouseFcγRIV (as well as several-fold enhanced binding to mouse FcγRII andmouse FcγRIII). It was thought from these results that the mouse FcγRsthat more significantly contribute to the efficient decrease of theantigen concentration in mouse plasma by administration of apH-dependent antigen-binding antibody, are mouse FcγRII and/or mouseFcγRIII. Specifically, it is thought that the plasma antigenconcentration can be more efficiently reduced in vivo by administeringinto a living organism a pH-dependent antigen-binding antibody withenhanced binding to mouse FcγRII and/or mouse FcγRIII.

(9-5) Preparation of Antigen-Binding Molecules Whose FcγR-BindingActivity is Greater than the Binding Activity of Native Human IgG FcRegion and which have Increased Human FcRn-Binding Activity Under anAcidic pH Range Condition

As described in Reference Example 8, when compared to human FcRntransgenic mice administered with Fv4-IgG1-F1022, the plasma antibodyretention is markedly improved in human FcRn transgenic miceadministered with Fv4-IgG1-F1093 resulting from increasing the humanFcRn-binding activity under an acidic pH range condition ofFv4-IgG1-F1022 in which the mouse FcγR-binding activity has beenincreased. Whether this effect is also observed in human FcRn transgenicmice administered with Fv4-IgG1-F1087 and Fv4-IgG1-F1182, and whetherthe same effect is observed in mice administered with variants whichhave increased human FcRn-binding activity under an acidic pH rangecondition by addition of an alteration distinct from the alterationassessed in Reference Example 8 were assessed as follows.

VH3-IgG1-F1180 (SEQ ID NO: 146) and VH3-IgG1-F1181 (SEQ ID NO: 147) wereprepared by substituting Leu for Met at position 428 and Ser for Asn atposition 434 (EU numbering) in the heavy chains VH3-IgG1-F1087 andVH3-IgG1-F1182, respectively, in order to increase their humanFcRn-binding activity of Fv4-IgG1-F1087 and Fv4-IgG1-F1182 under anacidic pH range condition. Furthermore, VH3-IgG1-F1412 (SEQ ID NO: 149)was prepared by substituting Ala for Asn at position 434 (EU numbering)in the heavy chain VH3-IgG1-F1087, in order to increase the humanFcRn-binding activity of Fv4-IgG1-F1087 under an acidic pH rangecondition. Fv4-IgG1-F1180, Fv4-IgG1-F1181, and Fv4-IgG1-F1412, whichcontain the above heavy chains and VL3-CK as the light chain, wereprepared using the method described in Reference Example 1.

(9-6) Improvement of Pharmacokinetics by Increasing the HumanFcRn-Binding Activity Under an Acidic pH Range Condition

In vivo infusion tests were carried out by administering Fv4-IgG1-F1180,Fv4-IgG1-F1181, and Fv4-IgG1-F1412 to human FcRn transgenic miceaccording to the same method as described in Reference Example 7 todetermine the soluble human IL-6 receptor concentrations in the plasmaof the mice. The results on the soluble human IL-6 receptorconcentrations in the plasma of the mouse groups administered withFv4-IgG1-F1087, Fv4-IgG1-F1180, Fv4-IgG1-F1412, and Fv4-IgG1 are shownin FIG. 23. The results on the soluble human IL-6 receptorconcentrations in the plasma of the mouse groups administered withFv4-IgG1-F1182, Fv4-IgG1-F1181, and Fv4-IgG1 are shown in FIG. 24.Meanwhile, the plasma antibody concentrations in the mouse groups weremeasured by the method described in Reference Example 8. The results onthe plasma antibody concentrations of Fv4-IgG1-F1087, Fv4-IgG1-F1180,Fv4-IgG1-F1412, and Fv4-IgG1 in the mouse groups are shown in FIG. 21;and the results on the plasma antibody concentrations of Fv4-IgG1-F1182,Fv4-IgG1-F1181, and Fv4-IgG1 are shown in FIG. 22.

It was confirmed that, as compared to the group of mice administeredwith Fv4-IgG1-F1182, the plasma antibody retention was improved in thegroup of mice administered with Fv4-IgG1-F1181 resulting from increasingthe human FcRn-binding activity of Fv4-IgG1-F1182 in an acidic pH range.Meanwhile, the soluble human IL-6 receptor concentration in the plasmaof the mouse groups administered with Fv4-IgG1-F1181 was comparable tothat in the group of mice administered with Fv4-IgG1-F1182. Whencompared to the mouse groups administered with Fv4-IgG1, the solublehuman IL-6 receptor concentration in the plasma was decreased in bothgroups.

On the other hand, as compared to the group of mice administered withFv4-IgG1-F1087, the plasma antibody retention was improved in bothgroups of mice administered with Fv4-IgG1-F1180 and Fv4-IgG1-F1412resulting from increasing the human FcRn-binding activity ofFv4-IgG1-F1087 in an acidic pH range, and surprisingly, the plasmaretention was improved up to a level comparable to that of the mousegroups administered with Fv4-IgG1. Furthermore, the sustainability ofthe effect of reducing the soluble human IL-6 receptor concentration inplasma was improved by the improvement of the plasma antibody retentionin the groups of administered mice. Specifically, in the groups ofadministered mice, the soluble human IL-6 receptor concentrations inplasma 14 days and 21 days after administration of Fv4-IgG1-F1180 andFv4-IgG1-F1412 were significantly reduced as compared to theconcentrations 14 days and 21 days after administration ofFv4-IgG1-F1087.

In view of the above, as for the groups of mice administered with thefour examples of antibodies, Fv4-IgG1-F1093, Fv4-IgG1-F1181,Fv4-IgG1-F1180, and Fv4-IgG1-F1412, it was demonstrated that the plasmaretention can be improved in a living organism administered with anantibody in which the human FcRn-binding activity under an acidic pHrange condition is enhanced in an antigen-binding molecule whoseFcγR-binding activity is higher than the binding activity of nativehuman IgG Fc region. It was also demonstrated that, in the livingorganism administered with the antigen-binding molecule, the plasmaretention is improved without deteriorating the effect of eliminatingantigens from the living organism, and rather, the antigen eliminationeffect can be sustained.

It was shown that the alteration that increases the human FcRn-bindingactivity under an acidic pH range condition can be achievable by notonly a method for substituting Leu for Met at position 428 and Ser forAsn at position 434 (EU numbering), but also a method for substitutingAla for Asn at position 434 (EU numbering). Thus, alterations used forincreasing the human FcRn-binding activity under an acidic pH rangecondition are not particularly limited, and include:

the method for substituting Leu for Met at position 428 and Ser for Asnat position 434 (EU numbering) in an IgG antibody (Nat. Biotechnol.(2010) 28, 157-159); the method for substituting Ala for Asn at position434 (Drug Metab. Dispos. (2010) 38 (4) 600-605); the method forsubstituting Tyr for Met at position 252, Thr for Ser at position 254,and Glu for Thr at position 256 (J. Biol. Chem. (2006) 281,23514-23524); the method for substituting Gln for Thr at position 250and Leu for Met at position 428 (J. Immunol. (2006) 176 (1), 346-356);and the method for substituting His for Asn at position 434 (Clin.Pharmcol. Ther. (2011) 89 (2) 283-290); and the alterations described inWO2010/106180, WO2010/045193, WO2009/058492, WO2008/022152,WO2006/050166; WO2006/053301, WO2006/031370, WO2005/123780,WO2005/047327, WO2005/037867, WO2004/035752, and WO2002/060919, etc.(9-7) Preparation of Antigen-Binding Molecules with Increased HumanFcRn-Binding Activity Under an Acidic pH Range Condition and SuppressedBinding to a Rheumatoid Factor

In recent years, an antibody molecule resulting from substituting Hisfor Asn at position 434 (EU numbering) in a humanized anti-CD4 antibodyto improve the plasma retention by increasing its human FcRn-bindingactivity under an acidic pH range condition, has been reported to bindto the rheumatoid factor (RF) (Clin. Pharmacol. Ther. (2011) 89 (2),283-290). This antibody has a human IgG1 Fc region and a substitution ofHis for Asn at position 434 (EU numbering) in the FcRn-binding site. Therheumatoid factor has been demonstrated to recognize and bind to thesubstituted portion.

As shown in (9-6), various alterations have been reported to increasethe human FcRn-binding activity under an acidic pH range condition.There is the possibility that the binding activity to the rheumatoidfactor that recognizes the site is increased by introducing suchalterations into the FcRn-binding site of the Fc region.

However, antigen-binding molecules that have increased humanFcRn-binding activity under an acidic pH range condition but do not havethe binding to the rheumatoid factor can be produced by introducing intothe site of the Fc region an alteration that reduces the rheumatoidfactor-binding activity alone without reducing the FcRn-binding activityunder an acidic pH range condition.

Such alterations used for reducing the rheumatoid factor-bindingactivity include alterations at positions 248-257, 305-314, 342-352,380-386, 388, 414-421, 423, 425-437, 439, and 441-444 (EU numbering),preferably those at positions 387, 422, 424, 426, 433, 436, 438, and 440(EU numbering), and particularly preferably, an alteration thatsubstitutes Glu or Ser for Val at position 422, an alteration thatsubstitutes Arg for Ser at position 424, an alteration that substitutesAsp for His at position 433, an alteration that substitutes Thr for Tyrat position 436, an alteration that substitutes Arg or Lys for Gln atposition 438, and an alteration that substitutes Glu or Asp for Ser atposition 440 (EU numbering). These alterations may be used alone or incombination.

Alternatively, it is possible to introduce N-type glycosylationsequences to reduce the rheumatoid factor-binding activity.Specifically, known N-type glycosylation sequences includeAsn-Xxx-Ser/Thr (Xxx represents an arbitrary amino acid other than Pro).This sequence can be introduced into the Fc region to add an N-typesugar chain, and the binding to RF can be inhibited by the sterichindrance of the N-type sugar chain. Alterations used for adding anN-type sugar chain preferably include an alteration that substitutes Asnfor Lys at position 248, an alteration that substitutes Asn for Ser atposition 424, an alteration that substitutes Asn for Tyr at position 436and Thr for Gln at position 438, and an alteration that substitutes ofAsn for Qln at position 438, according to EU numbering, particularlypreferably an alteration that substitutes Asn for Ser at position 424(EU numbering).

Reference Example 10 Effect of Eliminating Antigens from Plasma byAntigen-Binding Molecules Whose FcγR-Binding Activity is Higher than theBinding Activity of Native Mouse IgG Fc Region

(10-1) the Antigen Elimination Effect of Mouse Antibodies with IncreasedFcγR-Binding Activity

As described in Reference Examples 6 to 9, it was demonstrated that theelimination of soluble human IL-6 receptor from mouse plasma isaccelerated in the groups of human FcRn transgenic mice administeredwith antigen-binding molecules resulting from increasing the mouseFcγR-binding activity of antigen-binding molecules that have a humanantibody Fc region and the property of binding to human IL-6 receptor ina pH-dependent manner. Whether this effect is also achieved in normalmice having mouse FcRn that was administered with antigen-bindingmolecules that have a mouse antibody Fc region and the property ofbinding to human IL-6 receptor in a pH-dependent manner, was assessed asfollows.

(10-2) Preparation of Mouse Antibodies with Increased FcγR-BindingActivity

For a mouse IgG1 antibody having the property of binding to human IL-6receptor in a pH-dependent manner, the heavy chain VH3-mIgG1 (SEQ ID NO:150) and the light chain VL3-mk1 (SEQ ID NO: 151) were constructed usingthe method described in Reference Example 1. Meanwhile, to increase themouse FcγR-binding activity of VH3-mIgG1, VH3-mIgG1-mF44 (SEQ ID NO:152) was produced by substituting Asp for Ala at position 327 (EUnumbering). Likewise, VH3-mIgG1-mF46 (SEQ ID NO: 153) was produced bysubstituting Asp for Ser at position 239 and Asp for Ala at position327, according to EU numbering, in VH3-mIgG1. Fv4-mIgG1, Fv4-mIgG1-mF44,and Fv4-mIgG1-mF46, which contain VH3-mIgG1, VH3-mIgG1-mF44, andVH3-mIgG1-mF46, respectively, as the heavy chain, and VL3-mk1 as thelight chain, were prepared using the method described in ReferenceExample 1.

(10-3) Assessment of Mouse FcγR-Binding Activity

VH3/L (WT)-mIgG1, VH3/L (WT)-mIgG1-mF44, and VH3/L (WT)-mIgG1-mF46,which contain VH3-mIgG1, VH3-mIgG1-mF44, and VH3-mIgG1-mF46,respectively, as the heavy chain, and L (WT)-CK (SEQ ID NO: 5) as thelight chain, were prepared by the method described in ReferenceExample 1. These antibodies were assessed for their mouse FcγR-bindingactivity by the method described in Reference Example 2. The result isshown in Table 20. In addition, the ratio of the increase in the mouseFcγR-binding activity of each variant relative to the mIgG1 beforealteration is shown in Table 21. In the table, VH3/L (WT)-mIgG1 isabbreviated as mIgG1; VH3/L (WT)-mIgG1-mF44 is abbreviated as mF44; andVH3/L (WT)-mIgG1-mF46 is abbreviated as mF46.

TABLE 20 VARIANT KD (M) NAME mFcγRI mFcγRIIb mFcγRIII mFcγRIV mIgG1 NOTDETECTED 1.1E−07 2.1E−07 NOT DETECTED mF44 NOT DETECTED 8.9E−09 6.7E−09NOT DETECTED mF46 NOT DETECTED 1.2E−09 3.6E−09 NOT DETECTED

TABLE 21 VARIANT RATIO OF BINDING TO mIgG1 NAME mFcγRI mFcγRIIb mFcγRIIImFcγRIV mIgG1 NOT DETECTED 1.0 1.0 NOT DETECTED mF44 NOT DETECTED 11.931.0 NOT DETECTED mF46 NOT DETECTED 91.4 57.5 NOT DETECTED

The assessment result of Reference Example 9 showing that VH3/L(WT)-mIgG1 having the Fc region of native mouse IgG1 antibody only bindsto mouse FcγRIIb and mouse FcγRIII but not to mouse FcγRI and mouseFcγRIV, suggests that mouse FcγRs important for the reduction of antigenconcentration are mouse FcγRII and/or mouse FcγRIII. VH3/L(WT)-mIgG-mF44 and VH3/L (WT)-mIgG1-mF46 introduced with an alterationthat is thought to increase the FcγR-binding activity of VH3/L(WT)-mIgG1 was demonstrated to have increased binding activity to bothof mouse FcγRIIb and mouse FcγRIII.

(10-4) Assessment of the Effect to Reduce the Soluble Human IL-6Receptor Concentration in the Plasma of Normal Mice

The effect to eliminate soluble human IL-6 receptor from the plasma ofnormal mice administered with the anti-human IL-6 receptor antibodyFv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1mF46 was assessed as follows.

An animal model where the soluble human IL-6 receptor concentration ismaintained in a steady state in plasma was created by implanting aninfusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet) containing solublehuman IL-6 receptor under the skin on the back of normal mice (C57BL/6Jmouse, Charles River Japan). The in vivo kinetics of soluble human IL-6receptor after administration of the anti-human IL-6 receptor antibodywas assessed in the animal model. To suppress the production ofantibodies against soluble human IL-6 receptor, an anti-mouse CD4monoclonal antibody was administered once at 20 mg/kg into the tailvein. Then, an infusion pump containing 92.8 μg/ml soluble human IL-6receptor was subcutaneously implanted on the back of the mice. Threedays after implantation of the infusion pump, the anti-human IL-6receptor antibody was administered once at 1 mg/kg into the tail vein.The blood was collected from the mice 15 minutes, seven hours, one day,two days, four days, seven days, 14 days (or 15 days), and 21 days (or22 days) after administration of the anti-human IL-6 receptor antibody.Immediately thereafter, the collected blood was centrifuged at 15,000rpm and 4° C. for 15 minutes to prepare the plasma. The isolated plasmawas stored in a freezer set at −20° C. or below until use.

The soluble human IL-6 receptor concentrations in plasma were determinedby the method described in (7-1-2). The result is shown in FIG. 25.

Surprisingly, it was demonstrated that, in mice administered with mF44and mF46 introduced with an alteration to increase the binding activityof mIgG1 (native mouse IgG1) to mouse FcγRIIb and mouse FcγRIII, theplasma IL-6 receptor concentration was markedly reduced as compared tomice administered with mIgG1. In particular, even on day 21 afteradministration of mF44, the plasma IL-6 receptor concentration in themF44-administered group was reduced by about 6 times as compared to theplasma IL-6 receptor concentration in the group without antibodyadministration, and about 10 times as compared to the mIgG1-administeredgroup. On the other hand, on day seven after administration of mF46, theplasma IL-6 receptor concentration in the mF46-administered group wasmarkedly reduced by about 30 times as compared to the plasma IL-6receptor concentration in the group without antibody administration, andabout 50 times as compared to the mIgG1-administered group.

The above findings demonstrate that the elimination of soluble humanIL-6 receptor from plasma was also accelerated in mice administered withantibodies in which the mouse FcγR-binding activity of anantigen-binding molecule having the Fc regions of mouse IgG1 antibody isincreased, as with antibodies in which the mouse FcγR-binding activityof an antigen-binding molecule having the Fc region of human IgG1antibody is increased. Without being bound by a particular theory, thephenomenon observed as described above can be explained as follows.

When administered to mice, antibodies that bind to a soluble antigen ina pH-dependent manner and have increased FcγR-binding activity areactively incorporated mainly into cells expressing FcγR on the cellmembrane. The incorporated antibodies dissociate the soluble antigenunder an acidic pH condition in the endosome, and then recycled toplasma via FcRn. Thus, a factor that achieves the effect of eliminatingthe plasma soluble antigen of such an antibody is the FcγR-bindingactivity level of the antibody. Specifically, as the FcγR-bindingactivity is greater, the incorporation into FcγR-expressing cells occursmore actively, and this makes the elimination of soluble antigens fromplasma more rapid. Furthermore, as long as the FcγR-binding activity hasbeen increased, the effect can be assessed in the same manner regardlessof whether the Fc region contained in an antibody originates from humanor mouse IgG1. Specifically, the assessment can be achieved for an Fcregion of any animal species, such as any of human IgG1, human IgG2,human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, mouseIgG3, rat IgG, monkey IgG, and rabbit IgG, as long as the bindingactivity to the FcγR of the animal species to be administered has beenincreased.

Reference Example 11 The Antigen Elimination Effect by Antibodies withthe Binding Activity Increased in a FcγRIIb-Selective Manner

(11-1) the Antigen Elimination Effect of Antibodies in which theFcγRIIb-Binding Activity has been Selectively Increased

FcγRIII-deficient mice (B6.129P2-FcgrR3tm1Sjv/J mouse, JacksonLaboratories) express mouse FcγR1, mouse FcγRIIb, and mouse FcγRIV, butnot mouse FcγRIII. Meanwhile, Fc receptor γ chain-deficient mice (Fcer1gmouse, Taconic, Cell (1994) 76, 519-529) express mouse FcγRIIb alone,but not mouse FcγRI, mouse FcγRIII, and mouse FcγRIV.

As described in Reference Example 10, it was demonstrated that mF44 andmF46 with increased FcγR-binding activity of native mouse IgG1 showselectively enhanced binding to mouse FcγRIIb and mouse FcγRIII. It wasconceived that, using the selectively increased binding activity of theantibodies, the condition under which an antibody with selectivelyenhanced mouse FcγRIIb binding is administered can be mimicked byadministering mF44 and mF46 to mouse FcγRIII-deficient mice or Fcreceptor γ chain-deficient mice which do not express mouse FcγRIII.

(11-2) Assessment of the Antigen Elimination Effect by the MouseFcγRIIb-Selective Enhancement of Binding Using FcγRIII-Deficient Mice

The effect to eliminate soluble human IL-6 receptor from plasma inFcγRIII-deficient mice administered with the anti-human IL-6 receptorantibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1-mF46 was assessed bythe same method described in Reference Example 10. The soluble humanIL-6 receptor concentrations in the plasma of the mice were determinedby the method described in (7-1-2). The result is shown in FIG. 26.

Surprisingly, it was demonstrated that, the plasma IL-6 receptorconcentrations in FcγRIII-deficient mice administered with mF44 andmF46, which mimic the condition under which the mouse FcγRIIb-bindingactivity of mIgG1 (native mouse IgG1) is selectively increased, weremarkedly reduced as compared to the plasma IL-6 receptor concentrationin mice administered with mIgG1. In particular, the plasma IL-6 receptorconcentration of the mF44-administered group was reduced by about threetimes as compared to that of the mIgG1-administered group and theaccumulation of antigen concentration due to antibody administration wassuppressed. Meanwhile, on day three after administration, the plasmaIL-6 receptor concentration of the mF46-administered group was markedlyreduced by about six times as compared to the plasma IL-6 receptorconcentration of the group without antibody administration, and about 25times as compared to the plasma IL-6 receptor concentration of themIgG1-administered group. This result shows that, as the mouseFcγRIIb-binding activity of an anti-human IL-6 receptor antibody thatbinds to the antigen in a pH-dependent manner is greater, the IL-6receptor concentration can be reduced more in the plasma of miceadministered with the antibody.

(11-3) Assessment of the Antigen Elimination Effect by the SelectiveEnhancement of Mouse FcγRIIb Binding Using Fc Receptor γ Chain-DeficientMice

The effect to eliminate soluble human IL-6 receptor from the plasma ofFc receptor γ chain-deficient mice administered with the anti-human IL-6receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1mF46, wasassessed by the same method as described in Reference Example 10. Thesoluble human IL-6 receptor concentrations in the plasma of the micewere determined by the method described in (7-1-2). The result is shownin FIG. 27.

As with the case where mF44 and mF46 were administered toFcγRIII-deficient mice, the plasma IL-6 receptor concentration in Fcreceptor γ chain-deficient mice administered with mF44 and mF46, whichmimic the condition resulting from the selective increase in the mouseFcγRIIb-binding activity of mIgG1 (native mouse IgG1), was demonstratedto be markedly reduced as compared to the plasma IL-6 receptorconcentration in Fc receptor γ chain-deficient mice administered withmIgG1. In particular, the plasma IL-6 receptor concentration in themF44-administered group was reduced to about three times that in themIgG1-administered group, and the accumulation of antigen concentrationdue to antibody administration was suppressed. Meanwhile, on day threeafter administration, the plasma IL-6 receptor concentration in themF46-administered group was markedly reduced by about five times ascompared to that in the group without antibody administration, and about15 times as compared to that in the mIgG1-administered group.

The results described in (11-2) and (11-3) show that the soluble antigenconcentration in the plasma is markedly reduced in the groupadministered with an antibody that binds to a soluble antigen in apH-dependent manner and has selectively increased mouse FcγRIIb-bindingactivity.

Reference Example 12 The Antigen Elimination Effect of Antibodies withFcγRIII-Selective Binding Enhancement

(12-1) the Antigen Elimination Effect of Antibodies with SelectivelyEnhanced FcγRIII Binding

FcγRIIb-deficient mice (Fcgr2b (FcγRII) mouse, Taconic) (Nature (1996)379 (6563), 346-349) express mouse FcγRI, mouse FcγRIII, and mouseFcγRIV, but not mouse FcγRIIb. As described in Reference Example 10, itwas demonstrated that mF44 and mF46 resulting from increasing theFcγR-binding activity of native mouse IgG1 show selectively enhancedbinding to mouse FcγRIIb and mouse FcγRIII. It was conceived that, basedon the use of the selectively increased binding activity of theantibodies, the condition of administration of an antibody withselectively enhanced binding to mouse FcγRIII can be mimicked byadministering mF44 or mF46 to mouse FcγRIIb-deficient mice which do notexpress mouse FcγRIIb.

As described in Reference Example 11, the soluble antigen concentrationwas reduced in the plasma of FcγRIII-deficient mice, which mimic thecondition of administration of an antibody with selectively increasedmouse FcγRIIb-binding activity. Meanwhile, whether the soluble antigenconcentration is reduced in the plasma of FcγRIIb-deficient mice, whichmimic the condition of administration of an antibody with selectivelyincreased mouse FcγRIII-binding activity, was assessed by the testdescribed below.

(12-2) Assessment of the Antigen Elimination Effect by SelectiveEnhancement of Mouse FcγRIII Binding Using FcγRIIb-Deficient Mice

The effect to eliminate soluble human IL-6 receptor from the plasma ofFcγRIIb-deficient mice administered with the anti-human IL-6 receptorantibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1mF46, was assessed bythe same method as described in Reference Example 10. The soluble humanIL-6 receptor concentrations in plasma were determined by the methoddescribed in (7-1-2). The result is shown in FIG. 28.

Surprisingly, in the groups administered with mF44 and mF46, which mimicselective increase of the mouse FcγRIII-binding activity of mIgG1(native mouse IgG1), the plasma IL-6 receptor concentration was reduced,but the reduction was not as significant as that shown in ReferenceExample 11.

Without being bound by a particular theory, based on the resultsdescribed in Reference Examples 10, 11, and 12, the following discussionis possible. The elimination of soluble human IL-6 receptor from plasmawas found to be markedly accelerated in normal mice expressing bothmouse FcγRIIb and mouse FcγRIII that were administered with mF44 andmF46 with selectively increased binding activity of mIgG1 (native mouseIgG1) to mouse FcγRIIb and mouse FcγRIII. Furthermore, it was revealedthat, when mF44 and mF46 were administered to mice that express mouseFcγRIIb but not mouse FcγRIII (i.e., FcγRIII-deficient mice and Fcreceptor γ chain-deficient mice), the elimination of soluble human IL-6receptor from plasma was also accelerated markedly in the mice.Meanwhile, when mF44 and mF46 were administered to mice that expressmouse FcγRIII but not mouse FcγRIIb (i.e., FcγRII-deficient mice), theelimination of soluble human IL-6 receptor from plasma was not markedlyaccelerated in the mice.

From the above findings, it is thought that, the antibodies mF44 andmF46 in which the binding activity of mIgG1 (native mouse IgG1) to mouseFcγRIIb and mouse FcγRIII is selectively increased, are incorporatedinto FcγR-expressing cells mainly by mouse FcγRIIb, and thus the solubleantigen in the plasma that binds to the antibodies is eliminated.Meanwhile, the FcγRIII-mediated incorporation of antibody/antigencomplexes into FcγR-expressing cells is thought not to significantlycontribute to the elimination of the soluble antigen from plasma.

Furthermore, as shown in Reference Example 9, the plasma concentrationof soluble human IL-6 receptor was markedly reduced in mice administeredwith Fv4-IgG1-F1087 having increased binding activity to mouse FcγRIIband mouse FcγRIII, in particular. Meanwhile, the effect to eliminatesoluble human IL-6 receptor from the plasma of mice administered withFv4-IgG1-F1182 with increased binding activity to mouse FcγRI and mouseFcγRIV, in particular, was smaller than that of Fv4-IgG1-F1087.

Furthermore, as shown in Reference Example 7, in mice administered withFv4-IgG1-Fuc whose mouse FcγRIV-binding activity has been considerablyincreased by having sugar chains with low fucose content (Science (2005)310 (5753) 1510-1512), the plasma concentration of soluble human IL-6receptor was reduced as compared to that in mice administered withFv4-IgG1; however, the reduction effect was as small as about twice.Thus, mouse FcγRIV-mediated incorporation of antibodies intoFcγR-expressing cells is thought not to significantly contribute to theelimination of soluble antigens from plasma.

The above demonstrates that, in mice, of multiple mouse FcγRs, mouseFcγRIIb plays a major role in the incorporation of antibodies intoFcγR-expressing cells. Thus, mutations to be introduced into the mouseFcγ receptor-binding domain are particularly preferably, but are notparticularly limited to, mutations that enhance the binding to mouseFcγRIIb.

By this assessment using mice, it was demonstrated that, when anantigen-binding molecule that binds to a soluble antigen in apH-dependent manner and has increased FcγR-binding activity isadministered to accelerate the elimination of the soluble antigen fromplasma in the administered living organism, it is more preferable toincrease the FcγRIIb-binding activity of the antibody to beadministered. Specifically, it was revealed that, when administered tothe living organism, antigen-binding molecules that bind to a solubleantigen in a pH-dependent manner and have increased FcγRIIb-bindingactivity can effectively reduce the plasma concentration of the solubleantigen by accelerating the elimination of the soluble antigen fromplasma, and thus such antigen-binding molecules exhibit highly effectiveaction.

Reference Example 13 Assessment of the Platelet Aggregatory Ability ofAntibodies Containing an Fc Region Introduced with an ExistingAlteration that Enhances the FcγRIIb Binding

(13-1) Preparation of Antibodies Containing an Fc Region Introduced withan Existing Alteration that Enhances the FcγRIIb Binding

As described in Reference Example 12, antigens can be efficientlyeliminated from the plasma of the living organism by administeringantibodies with selectively increased FcγRIIb-binding activity to theliving organism. Furthermore, the administration of antibodiescontaining an Fc region with selectively increased FcγRIIb-bindingactivity is thought to be preferred from the viewpoint of safety andside effects in the living organism administered with such antibodies.

However, the mouse FcγRIIb binding and mouse FcγRIII binding are bothenhanced in mF44 and mF46, and thus the binding enhancement is notselective for mouse FcγRIIb. Since the homology between mouse FcγRIIband mouse FcγRIII is high, it would be difficult to find an alterationthat enhances the mouse FcγRIIb-selective binding while distinguishingthe two. Moreover, there is no previous report on Fc regions withselectively enhanced mouse FcγRIIb binding. In addition, the homologybetween human FcγRIIb and human FcγRIIa (allotypes H131 and R131) isalso known to be high. Furthermore, it has been reported that antibodieswith enhanced FcγRIIa binding have increased platelet aggregatoryactivity, and can increase the risk of developing thrombosis in theliving organism administered with them (Meyer et al., (J. Thromb.Haemost. (2009), 7 (1), 171-181), Robles-Carrillo et al., (J. Immunol.(2010), 185 (3), 1577-1583)). Thus, whether antibodies with enhancedFcγRIIa binding have increased platelet aggregatory activity wasassessed as follows.

(13-2) Assessment of the Human FcγR-Binding Activity of AntibodiesContaining an Fc Region Introduced with an Existing Alteration thatEnhances the FcγRIIb Binding

Antibodies containing an Fc region introduced with an existingalteration that enhances the human FcγRIIb binding were analyzed fortheir affinity for human FcγRIa, R-type and H-type FcγRIIa, FcγRIIb, andFcγRIIIa by the following procedure. An H chain was constructed to have,as the antibody H chain variable region, the antibody variable regionIL6R-H (SEQ ID NO: 154) against human IL-6 receptor which is disclosedin WO2009/125825, and as the antibody H chain constant region, IL6R-G1d(SEQ ID NO: 156) that has G1d resulting from removing the C-terminal Glyand Lys from human IgG1. Then, IL6R-G1d-v3 (SEQ ID NO: 157) wasconstructed by altering the Fc region of IL6R-G1d by the substitution ofGlu for Ser at position 267 (EU numbering) and Phe for Leu at position328 (EU numbering), as described in Seung et al., (Mol. Immunol. (2008)45, 3926-3933). IL6R-L (SEQ ID NO: 155) which is the L chain ofanti-human IL-6 receptor antibody was used as a common antibody L chain,and expressed in combination with respective H chains according to themethod described in Reference Example 1, and the resulting antibodieswere purified. Hereinafter, antibodies containing IL6R-G1d andIL6R-G1d-v3 as the heavy chain are referred to as IgG1 and IgG1-v3,respectively.

Then, the interaction between FcγR and the above antibodies waskinetically analyzed using Biacore T100 (GE Healthcare). The assay forthe interaction was carried out at 25° C. using HBS-EP+(GE Healthcare)as a running buffer. The chip used was a Series S Sencor Chip CM5 (GEHealthcare) immobilized with Protein A by an amine coupling method. EachFcγR diluted with the running buffer was allowed to interact with theantibodies of interest captured onto the chip to measure the binding ofthe antibodies to each FcγR. After measurement, 10 mM glycine-HCl (pH1.5) was reacted to the chip to wash off the captured antibodies torepeatedly use the regenerated chip. A sensorgram obtained as a resultof the measurement was analyzed using 1:1 Langmuir binding model withBiacore Evaluation Software, and binding rate constant ka (L/mol/s) anddissociation rate constant kd (1/s) were calculated, and thedissociation constant KD (mol/l) was calculated from these values. TheKD values of IgG1 and IgG1-v3 to each FcγR are shown in Table 22 (the KDvalues of each antibody to each FcγR), while the relative KD values ofIgG1-v3, which are obtained by dividing KD of IgG1 to each FcγR by KD ofIgG1-v3 to each FcγR, are shown in Table 23.

TABLE 22 KD (M) ANTIBODY FcγRIa FcγRIIaR FcγRIIaH FcγRIIb FcγRIIIa IgG13.4E−10 1.2E−06 7.7E−07 5.3E−06 3.1E−06 IgG1-v3 1.9E−10 2.3E−09 1.5E−061.3E−08 8.8E−06

TABLE 23 FcγRIa FcγRIIaR FcγRIIaH FcγRIIb FcγRIIIa KD VALUE 1.8 522 0.51408 0.35 RATIO

The above results show that, as compared to the antibody containing theFc region of IgG1, the antibody containing an altered Fc region (Mol.Immunol. (2008) 45, 3926-3933) with the substitution of Glu for Ser atposition 267 and Phe for Leu at position 328 (EU numbering) in the Fcregion of IgG1, its affinity for FcγRIIb has been increased by 408times; its affinity for H-type FcγRIIa has been reduced to 0.51 times;and its affinity for R-type FcγRIIa has been increased by 522 times.

(13-3) Assessment of the Ability to Aggregate Platelets

Next, whether the increased/reduced FcγRIIa affinity of the antibodycontaining the Fc region with the substitution of Glu for Ser atposition 267 and Phe for Leu at position 328 (EU numbering) in the Fcregion of IgG1 changes the platelet aggregatory ability, was assessedusing platelets derived from donors with H-type or R-type FcγRIIa. Theantibody comprising as the light chain omalizumab_VL-CK (SEQ ID NO: 159)and omalizumab_VH-G1d (SEQ ID NO: 158) that contains the heavy chainvariable region of hIgG1 antibody (human IgG1 constant region) thatbinds to IgE and the G1d heavy chain constant region, was constructedusing the method described in Reference Example 1. Furthermore,omalizumab_VH-G1d-v3 (SEQ ID NO: 160) was constructed by substitutingGlu for Ser at position 267 and Phe for Leu at position 328 (EUnumbering) in omalizumab_VH-G1d. Omalizumab-G1d-v3, which containsomalizumab_VH-G1d-v3 as the heavy chain and omalizumab_VL-CK as thelight chain, was prepared using the method described in ReferenceExample 1. This antibody was assessed for the platelet aggregatoryability.

Platelet aggregation was assayed using the platelet aggregometer HEMATRACER 712 (LMS Co.). First, about 50 ml of whole blood was collected ata fixed amount into 4.5-ml evacuated blood collection tubes containing0.5 ml of 3.8% sodium citrate, and this was centrifuged at 200 g for 15minutes. The resultant supernatant was collected and used asplatelet-rich plasma (PRP). After PRP was washed with buffer A (137 mMNaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.42 mM NaH₂PO₄, 2 mM MgCl₂, 5 mM HEPES,5.55 mM dextrose, 1.5 U/ml apyrase, 0.35% BSA), the buffer was replacedwith buffer B (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.42 mM NaH₂PO₄, 2mM MgCl₂, 5 mM HEPES, 5.55 mM dextrose, 2 mM CaCl₂, 0.35% BSA). Thisyielded washed platelets at a density of about 300,000/μl. 156 μl of thewashed platelets was aliquoted into assay cuvettes containing a stir barin the platelet aggregometer. The platelets were stirred at 1000 rpmwith the stir bar in the cuvettes maintained at 37.0° C. in the plateletaggregometer. 44 μl of the immune complex of omalizumab-G1d-v3 and IgEat a molar ratio of 1:1, prepared at final concentrations of 600 μg/mland 686 μg/ml, respectively, was added to the cuvettes. The plateletswere reacted with the immune complex for five minutes. Then, at aconcentration that does not allow secondary platelet aggregation,adenosine diphosphate (ADP, SIGMA) was added to the reaction mixture totest whether the aggregation is enhanced.

The result of platelet aggregation for each donor with an FcγRIIapolymorphic form (H/H or R/H) obtained from the above assay is shown inFIGS. 29 and 30. The result in FIG. 29 shows that platelet aggregationis enhanced when the immune complex is added to the platelets of a donorwith the FcγRIIa polymorphic form (R/H). Meanwhile, as shown in FIG. 30,platelet aggregation was not enhanced when the immune complex is addedto the platelets of a donor with the FcγRIIa polymorphic form (H/H).

Next, platelet activation was assessed using activation markers.Platelet activation can be measured based on the increased expression ofan activation marker such as CD62p (p-selectin) or active integrin onthe platelet membrane surface. 2.3 μl of the immune complex was added to7.7 μl of the washed platelets prepared by the method described above.After five minutes of reaction at room temperature, activation wasinduced by adding ADP at a final concentration of 30 μM, and whether theimmune complex enhances the ADP-dependent activation was assessed. Asample added with phosphate buffer (pH 7.4) (Gibco), instead of theimmune complex, was used as a negative control. Staining was performedby adding, to each post-reaction sample, PE-labeled anti-CD62 antibody(BECTON DICKINSON), PerCP-labeled anti-CD61 antibody, and FITC-labeledPAC-1 antibody (BD bioscience). Fluorescence intensity for each stainwas measured using a flow cytometer (FACS CantoII, BD bioscience).

The result on CD62p expression, obtained by the above assay method, isshown in FIG. 31. The result on the activated integrin expression isshown in FIG. 32. The washed platelets used were obtained from a healthyperson with the FcγRIIa polymorphic form R/H. The expression of bothCD62p and active integrin on platelet membrane surface, which is inducedby ADP stimulation, was enhanced in the presence of the immune complex.

The above results demonstrate that the antibody having the Fc regionintroduced with an existing alteration that enhances the human FcγRIIbbinding, which is the substitution of Glu for Ser at position 267 andPhe for Leu at position 328 (EU numbering) in the Fc region of IgG1,promotes the aggregation of platelets with the FcγRIIa allotype in whichthe amino acid at position 131 is R, as compared to platelets with theFcγRIIa polymorphic form in which the amino acid at position 131 is H.That is, it was suggested that the risk of developing thrombosis due toplatelet aggregation can be increased when an antibody containing an Fcregion introduced with an existing alteration that enhances the humanFcγRIIb binding is administered to humans having R-type FcγRIIa. It wasshown that the antigen-binding molecules containing an Fc region of thepresent invention that enhances the FcγRIIb binding more selectively notonly improves the antigen retention in plasma, but also possibly solvesthe above problems. Thus, the usefulness of the antigen-bindingmolecules of the present invention is obvious.

Reference Example 14 Comprehensive Analysis of FcγRIIb Binding ofVariants Introduced with an Alteration at the Hinge Portion in Additionto the P238D Alteration

In an Fc produced by substituting Pro at position 238 (EU numbering)with Asp in a naturally-occurring human IgG1, an anticipatedcombinatorial effect could not be obtained even by combining it withanother alteration predicted to further increase FcγRIIb binding fromthe analysis of naturally-occurring antibodies. Therefore, in order tofind variants that further enhance FcγRIIb binding, alterations werecomprehensively introduced into the altered Fc produced by substitutingPro at position 238 (EU numbering) with Asp. IL6R-F11 (SEQ ID NO: 161)was produced by introducing an alteration of substituting Met atposition 252 (EU numbering) with Tyr and an alteration of substitutingAsn at position 434 (EU numbering) with Tyr in IL6R-G1d (SEQ ID NO: 156)which was used as the antibody H chain. Furthermore, IL6R-F652 (SEQ IDNO: 162) was prepared by introducing an alteration of substituting Proat position 238 (EU numbering) with Asp into IL6R-F11. Expressionplasmids containing an antibody H chain sequence were prepared for eachof the antibody H chain sequences produced by substituting the regionnear the residue at position 238 (EU numbering) (positions 234 to 237,and 239 (EU numbering)) in L6R-F652 each with 18 amino acids excludingthe original amino acid and Cys. IL6R-L (SEQ ID NO: 155) was utilized asan antibody L chain. These variants were expressed and purified by themethod of Reference Example 1. These Fc variants are called PD variants.Interactions of each PD variant with FcγRIIa type R (allotype R131) andFcγRIIb were comprehensively evaluated by the method of ReferenceExample 2.

A figure that shows the results of analyzing the interaction with therespective FcγRs was produced according to the following method. Thevalue obtained by dividing the value for the amount of binding of eachPD variant to each FcγR by the value for the amount of FcγR binding ofthe pre-altered antibody which is used as the control (IL6R-F652/IL6R-L,which has an alteration of substituting Pro at position 238 (EUnumbering) with Asp) and then multiplying the result by 100, was shownas the relative binding activity value of each PD variant to each FcγR.The horizontal axis shows relative values of the FcγRIIb-bindingactivity of each PD variant, and the vertical axis shows relative valuesof the FcγRIIa type R-binding activity of each PD variant (FIG. 33).

As a result, it was found that the FcγRIIb binding of eleven types ofalterations were enhanced compared with the antibody before introducingalterations, and they have the effects of maintaining or enhancingFcγRIIa type R-binding. The activities of these eleven variants to bindFcγRIIb and FcγRIIa type R are summarized in Table 24. In the table,alteration refers to the alteration introduced into IL6R-F11 (SEQ ID NO:161).

TABLE 24 RELATIVE RELATIVE BINDING BINDING ACTIVITY ACTIVITY VARIANTNAME ALTERATION TO FcγRIIb TO FcγRIIaR IL6R-F652/IL6R-L P238D 100 100IL6R-PD042/IL6R-L P238D/L234W 106 240 IL6R-PD043/IL6R-L P238D/L234Y 112175 IL6R-PD079/IL6R-L P238D/G237A 101 138 IL6R-PD080/IL6R-L P238D/G237D127 222 IL6R-PD081/IL6R-L P238D/G237E 101 117 IL6R-PD082/IL6R-LP238D/G237F 108 380 IL6R-PD086/IL6R-L P238D/G237L 112 268IL6R-PD087/IL6R-L P238D/G237M 109 196 IL6R-PD094/IL6R-L P238D/G237W 122593 IL6R-PD095/IL6R-L P238D/G237Y 124 543 IL6R-PD097/IL6R-L P238D/S239D139 844

FIG. 34 shows relative values for the FcγRIIb-binding activity of avariant obtained by additionally introducing the above elevenalterations into a variant carrying the P238D alteration, and relativevalues for the FcγRIIb-binding activity of a variant obtained byintroducing the alterations into an Fc that does not contain the P238D.These eleven alterations enhanced the amount of FcγRIIb binding comparedwith before introduction when they were further introduced into theP238D variant. On the contrary, the effect of lowering FcγRIIb bindingwas observed for eight of those alterations except G237F, G237W, andS239D, when they were introduced into the variant that does not containP238D (data not shown).

These results showed that, based on the effects of introducingalterations into a naturally-occurring IgG1, it is difficult to predictthe effects of combining and introducing the same alterations into thevariant containing the P238D alteration. In other words, it would nothave been possible to discover these eight alterations identified thistime without this investigation that the same alterations are combinedand introduced into the variant containing the P238D alteration.

The results of measuring KD values of the variants indicated in Table 24for FcγRIa, FcγRIIaR (allotype R131), FcγRIIaH (allotype H131), FcγRIIb,and FcγRIIIaV (allotype V158) by the method of Reference Example 2 aresummarized in Table 25. In the table, alteration refers to thealteration introduced into IL6R-F11 (SEQ ID NO: 161). The template usedfor producing IL6R-F11, IL6R-G1d/IL6R-L, is indicated with an asterisk(*). Furthermore, KD (IIaR)/KD (IIb) and KD (IIaH)/KD (IIb) in the tablerespectively show the value obtained by dividing the KD value of eachvariant for FcγRIIaR by the KD value of each variant for FcγRIIb, andthe value obtained by dividing the KD value of each variant for FcγRIIaHby the KD value of each variant for FcγRIIb. KD (IIb) of the parentpolypeptide/KD (IIb) of the variant refers to a value obtained bydividing the KD value of the parent polypeptide for FcγRIIb by the KDvalue of each variant for FcγRIIb. In addition, Table 25 shows KD valuesfor the stronger of the FcγRIIaR- and FcγRIIaH-binding activities ofeach variant/KD values for the stronger of the FcγRIIaR- andFcγRIIaH-binding activities of the parent polypeptide. Here, parentpolypeptide refers to a variant which has IL6R-F11 (SEQ ID NO: 161) asthe H chain. It was determined that due to weak binding of FcγR to IgG,it was impossible to accurately analyze by kinetic analysis, and thusthe gray-filled cells in Table 25 show values calculated by usingEquation 2 of Reference Example 2.

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

Table 25 shows that all variants improved their affinity for FcγRIIb incomparison with IL6R-F11, and the range of improvement was 1.9 fold to5.0 fold. The ratio of KD value of each variant for FcγRIIaR/KD value ofeach variant for FcγRIIb, and the ratio of KD value of each variant forFcγRIIaH/KD value of each variant for FcγRIIb represent anFcγRIIb-binding activity relative to the FcγRIIaR-binding activity andFcγRIIaH-binding activity, respectively. That is, these values show thedegree of binding selectivity of each variant for FcγRIIb, and a largervalue indicates a higher binding selectivity for FcγRIIb. For the parentpolypeptide IL6R-F11/IL6R-L, the ratio of KD value for FcγRIIaR/KD valuefor FcγRIIb and the ratio of KD value for FcγRIIaH/KD value for FcγRIIbare both 0.7, and accordingly all variants in Table 25 showedimprovement of binding selectivity for FcγRIIb in comparison with theparent polypeptide. When the KD value for the stronger of the FcγRIIaR-and FcγRIIaH-binding activities of a variant/KD value for the strongerof the FcγRIIaR- and FcγRIIaH-binding activities of the parentpolypeptide is 1 or more, this means that the stronger of the FcγRIIaR-and FcγRIIaH-binding activities of a variant has equivalent or reducedbinding compared with the binding by the stronger of the FcγRIIaR- andFcγRIIaH-binding activities of the parent polypeptide. Since this valuewas 0.7 to 5.0 for the variants obtained this time, one may say thatbinding by the stronger of the FcγRIIaR- and FcγRIIaH-binding activitiesof the variants obtained this time was nearly the same or decreased incomparison with the parent polypeptide. These results showed thatcompared with the parent polypeptide, the variants obtained this timehave maintained or decreased binding activities to FcγRIIa type R andtype H and enhanced binding activity to FcγRIIb, and thus have improvedselectivity for FcγRIIb. Furthermore, compared with IL6R-F11, allvariants had lower affinity to FcγRIa and FcγRIIIaV.

TABLE 25

Reference Example 15 X-Ray Crystal Structure Analysis of a ComplexFormed Between an Fc Containing P238D and an Extracellular Region ofFcγRIIb

As indicated earlier in Reference Example 14, even though an alterationthat is predicted from the analysis of naturally-occurring IgG1antibodies to improve FcγRIIb-binding activity or selectivity forFcγRIIb is introduced into an Fc containing P238D, the FcγRIIb-bindingactivity was found to decrease, and the reason for this may be that thestructure at the interacting interface between Fc and FcγRIIb is changeddue to introduction of P238D. Therefore, to pursue the reason for thisphenomena, the three-dimensional structure of the complex formed betweenan IgG1 Fc containing the P238D mutation (hereinafter, referred to as Fc(P238D)) and the extracellular region of FcγRIIb was elucidated by X-raycrystal structure analysis, and this was compared to thethree-dimensional structure of the complex formed between the Fc of anaturally-occurring IgG1 (hereinafter, referred to as Fc (WT)) and theextracellular region of FcγRIIb, and the binding modes were compared.Multiple reports have been made on the three-dimensional structure of acomplex formed between an Fc and an FcγR extracellular region; and thethree-dimensional structures of the Fc (WT)/FcγRIIIb extracellularregion complex (Nature (2000) 400, 267-273; J. Biol. Chem. (2011) 276,16469-16477), the Fc (WT)/FcγRIIIa extracellular region complex (Proc.Natl. Acad. Sci. USA (2011) 108, 12669-126674), and the Fc (WT)/FcγRIIaextracellular region complex (J. Immunol. (2011) 187, 3208-3217) havebeen analyzed. While the three-dimensional structure of the Fc(WT)/FcγRIIb extracellular region complex has not been analyzed, thethree-dimensional structure of a complex formed with Fc (WT) is knownfor FcγRIIa, and the extracellular regions of FcγRIIa and FcγRIIb match93% in amino acid sequence and have very high homology. Thus, thethree-dimensional structure of the Fc (WT)/FcγRIIb extracellular regioncomplex was predicted by modeling using the crystal structure of the Fc(WT)/FcγRIIa extracellular region complex.

The three-dimensional structure of the Fc (P238D)/FcγRIIb extracellularregion complex was determined by X-ray crystal structure analysis at 2.6Å resolution. The structure obtained as a result of this analysis isshown in FIG. 35. The FcγRIIb extracellular region is bound between twoFc CH2 domains, and this was similar to the three-dimensional structuresof complexes formed between Fc (WT) and the respective extracellularregion of FcγRIIIa, FcγRIIIb, or FcγRIIa analyzed so far. Next, fordetailed comparison, the crystal structure of the Fc (P238D)/FcγRIIbextracellular region complex and the model structure of the Fc(WT)/FcγRIIb extracellular region complex were superimposed by the leastsquares fitting based on the Cα atom pair distances with respect to theFcγRIIb extracellular region and the Fc CH2 domain A (FIG. 36). In thatcase, the degree of overlap between Fc CH2 domains B was notsatisfactory, and conformational differences were found in this portion.Furthermore, using the crystal structure of the Fc (P238D)/FcγRIIbextracellular region complex and the model structure of the Fc(WT)/FcγRIIb extracellular region complex, pairs of atoms that have adistance of 3.7 Å or less between the FcγRIIb extracellular region andFc CH2 domain B were extracted and compared in order to compare theinteratomic interaction between FcγRIIb and Fc (WT) CH2 domain B withthe interatomic interaction between FcγRIIb and Fc (P238D) CH2 domain B.As shown in Table 26, the interatomic interactions between Fc CH2 domainB and FcγRIIb in Fc (P238D) and Fc (WT) did not match.

TABLE 26 Fc(P648D) CH2 DOMAIN B Fc(WT) CH2 DOMAIN B INTERACTION PARTNERINTERACTION PARTNER FcγRIIb ATOM (DISTANCE BETWEEN ATOMS, Å) (DISTANCEBETWEEN ATOMS, Å) Val 116 CG2 Asp 265 OD2 (3.47) Gly 237 O (3.65) Ser126 OG Ser 298 N (3.31) Ser 298 CB (3.32) Tyr 296 O (3.05) Lys 128 CASer 298 OG (3.50) Phe 129 CB Ser 298 O (3.36) Phe 129 CD2 Asn 297 CB(3.50) Asn 297 CG (3.43) Lys 128 C Ser 298 OG (3.47) Phe 129 N Ser 298OG (3.30) Phe 129 O Ser 267 OG (3.54) Arg 131 CB Val 266 O (3.02) Arg131 CG Val 266 O (3.22) Arg 131 CD Val 266 CG1 (3.45) Val 266 C (3.55)Val 266 O (3.10) Arg 131 NE Ala 327 O (3.60) Val 266 C (3.66) Val 266 O(3.01) Val 266 N (3.49) Arg 131 CZ Asp 270 CG (3.64) Val 266 N (3.13)Asp 270 OD2 (3.22) Asp 270 OD1 (3.27) Ala 327 CB (3.63) Arg 131 NH1 Asp270 CG (3.19) Val 266 CG1 (3.47) Asp 270 OD2 (2.83) Val 266 N (3.43) Asp270 OD1 (2.99) Thr 299 OG1 (3.66) Ser 267 CB (3.56) Ser 298 O (3.11) Arg131 NH2 Asp 270 CG (3.20) Asp 270 OD2 (2.80) Asp 265 CA (3.16) Asp 270OD1 (2.87) Val 266 N (3.37) Ala 327 CB (3.66) Tyr 157 CE1 Leu 234 CG(3.64) Leu 234 CD1 (3.61) Tyr 157 OH Gly 236 O (3.62) Leu 234 CA (3.48)Leu 234 CG (3.45)

Furthermore, the detailed structures around P238D were compared bysuperposing the X-ray crystal structure of Fc (P238D)/FcγRIIbextracellular region complex on the model structure of the Fc(WT)/FcγRIIb extracellular region complex using the least squares methodbased on the Cα atomic distance between Fc CH2 domains A and B alone. Asthe position of the amino acid residue at position 238 (EU numbering),i.e., a mutagenesis position of Fc (P238D), is altered from Fc (WT), theloop structure around the amino acid residue at position 238 followingthe hinge region is found to be different between Fc (P238D) and Fc (WT)(FIG. 37). Pro at position 238 (EU numbering) is originally locatedinside Fc (WT), forming a hydrophobic core with residues around position238. However, if Pro at position 238 (EU numbering) is altered to highlyhydrophilic and charged Asp, the presence of the altered Asp residue ina hydrophobic core is energetically disadvantageous in terms ofdesolvation. Therefore, in Fc (P238D), to cancel this energeticallydisadvantageous situation, the amino acid residue at position 238 (EUnumbering) may have changed its orientation to face the solvent side,and this may have caused the change in the loop structure near the aminoacid residue at position 238. Furthermore, since this loop is not farfrom the hinge region crosslinked by an S—S bond, its structural changewill not be limited to a local change, and will affect the relativepositioning of the Fc CH2 domain A and domain B. As a result, theinteratomic interactions between FcγRIIb and Fc CH2 domain B have beenchanged. Therefore, predicted effects could not be observed whenalterations that improve selectivity and binding activity towardsFcγRIIb in a naturally-occurring IgG were combined with an Fc containingthe P238D alteration.

Furthermore, as a result of structural changes due to introduction ofP238D in Fc CH2 domain A, a hydrogen bond has been found between themain chain of Gly at position 237 (EU numbering), which is adjacent toP238D mutated, and Tyr at position 160 in FcγRIIb (FIG. 38). The residuein FcγRIIa that corresponds to this Tyr 160 is Phe; and when the bindingis to FcγRIIa, this hydrogen bond is not formed. Considering that theamino acid at position 160 is one of the few differences between FcγRIIaand FcγRIIb at the interface of interaction with Fc, the presence ofthis hydrogen bond which is specific to FcγRIIb is presumed to have ledto improvement of FcγRIIb-binding activity and decrease ofFcγRIIa-binding activity in Fc (P238D), and improvement of itsselectivity. Furthermore, in Fc CH2 domain B, an electrostaticinteraction is observed between Asp at position 270 (EU numbering) andArg at position 131 in FcγRIIb (FIG. 39). In FcγRIIa type H, which isone of the allotypes of FcγRIIa, the residue corresponding to Arg atposition 131 of FcγRIIb is His, and therefore cannot form thiselectrostatic interaction. This can explain why the Fc (P238D)-bindingactivity is lowered in FcγRIIa type H compared with FcγRIIa type R.Observations based on such results of X-ray crystal structure analysisshowed that the change of the loop structure beside P238D due to P238Dintroduction and the accompanying change in the relative domainpositioning could cause formation of new interactions which is not foundin the binding of the naturally-occurring IgG and FcγR, and this couldlead to a selective binding profile of P238D variants for FcγRIIb.

[Expression and Purification of Fc (P238D)]

An Fc containing the P238D alteration was prepared as follows. First,Cys at position 220 (EU numbering) of hIL6R-IgG1-v1 (SEQ ID NO: 163) wassubstituted with Ser. Then, genetic sequence of Fc (P238D) from Glu atposition 236 (EU numbering) to its C terminal was cloned by PCR. Usingthis cloned genetic sequence, production of expression vectors, andexpression and purification of Fc (P238D) were carried out according tothe method of Reference Example 1. Cys at position 220 (EU numbering)forms a disulfide bond with Cys of the L chain in general IgG1. The Lchain is not co-expressed when Fc alone is prepared, and therefore, theCys residue was substituted with Ser to avoid formation of unnecessarydisulfide bonds.

[Expression and Purification of the FcγRIIb Extracellular Region]

The FcγRIIb extracellular region was prepared according to the method ofReference Example 2.

[Purification of the Fc (P238D)/FcγRIIb Extracellular Region Complex]

To 2 mg of the FcγRIIb extracellular region sample obtained for use incrystallization, 0.29 mg of Endo F1 (Protein Science (1996) δ:2617-2622) expressed and purified from Escherichia coli as a glutathioneS-transferase fusion protein was added. This was allowed to remain atroom temperature for three days in 0.1 M Bis-Tris buffer at pH 6.5, andthe N-linked oligosaccharide was cleaved, except for N-acetylglucosaminedirectly bound to Asn of the FcγRIIb extracellular region. Next, theFcγRIIb extracellular region sample subjected to carbohydrate cleavagetreatment, which was concentrated by ultrafiltration with 5000 MWCO, waspurified by gel filtration chromatography (Superdex200 10/300) using acolumn equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl.Furthermore, to the obtained carbohydrate-cleaved FcγRIIb extracellularregion fraction, Fc (P238D) was added so that the molar ratio of theFcγRIIb extracellular region would be present in slight excess. Themixture concentrated by ultrafiltration with 10,000 MWCO was subjectedto purification by gel filtration chromatography (Superdex200 10/300)using a column equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 MNaCl. Thus, a sample of the Fc (P238D)/FcγRIIb extracellular regioncomplex was obtained.

[Crystallization of the Fc (P238D)/FcγRIIb Extracellular Region Complex]

Using the sample of the Fc (P238D)/FcγRIIb extracellular region complexwhich was concentrated to approximately 10 mg/mL by ultrafiltration with10,000 MWCO, crystallization of the complex was carried out by thesitting drop vapor diffusion method. Hydra II Plus One (MATRIX) was usedfor crystallization; and for a reservoir solution containing 100 mMBis-Tris pH 6.5, 17% PEG3350, 0.2 M ammonium acetate, and 2.7% (w/v)D-Galactose, a crystallization drop was produced by mixing at a ratio ofreservoir solution:crystallization sample=0.2 μL: 0.2 μL. Thecrystallization drop after sealing was allowed to remain at 20° C., andthus thin plate-like crystals were obtained.

[Measurement of X-Ray Diffraction Data from an Fc (P238D)/FcγRIIbExtracellular Region Complex Crystal]

One of the obtained single crystals of the Fc (P238D)/FcγRIIbextracellular region complex was soaked into a solution of 100 mMBis-Tris pH 6.5, 20% PEG3350, ammonium acetate, 2.7% (w/v) D-Galactose,22.5% (v/v) ethylene glycol. The single crystal was fished out of thesolution using a pin with attached tiny nylon loop, and frozen in liquidnitrogen. Then, the X-ray diffraction data of the crystal was measuredat synchrotron radiation facility Photon Factory BL-1A in High EnergyAccelerator Research Organization. During the measurement, the crystalwas constantly placed in a nitrogen stream at −178° C. to maintain in afrozen state, and a total of 225 X-ray diffraction images were collectedusing Quantum 270 CCD detector (ADSC) attached to a beam line withrotating the crystal 0.8° at a time. Determination of cell parameters,indexing of diffraction spots, and diffraction data processing from theobtained diffraction images were performed using the Xia2 program (CCP4Software Suite), XDS Package (Walfgang Kabsch) and Scala (CCP4 SoftwareSuite); and finally, diffraction intensity data of the crystal up to2.46 Å resolution was obtained. The crystal belongs to the space groupP21, and has the following cell parameters; a=48.85 Å, b=76.01 Å,c=115.09 Å, α=90°, β=100.70°, γ=90°.

[X-Ray Crystal Structure Analysis of the Fc (P238D)/FcγRIIbExtracellular Region Complex]

Crystal structure of the Fc (P238D)/FcγRIIb extracellular region complexwas determined by the molecular replacement method using the programPhaser (CCP4 Software Suite). From the size of the obtained crystallattice and the molecular weight of the Fc (P238D)/FcγRIIb extracellularregion complex, the number of complexes in the asymmetric unit waspredicted to be one. From the structural coordinates of PDB code: 3SGJwhich is the crystal structure of the Fc (WT)/FcγRIIIa extracellularregion complex, the amino acid residue portions of the A chain positions239-340 and the B chain positions 239-340 were taken out as separatecoordinates, and they were set respectively as models for searching theFc CH2 domains. The amino acid residue portions of the A chain positions341-444 and the B chain positions 341-443 were taken out as a single setof coordinates from the same structural coordinates of PDB code: 3SGJ;and this was set as a model for searching the Fc CH3 domains. Finally,from the structural coordinates of PDB code: 2FCB which is a crystalstructure of the FcγRIIb extracellular region, the amino acid residueportions of the A chain positions 6-178 was taken out and set as a modelfor searching the FcγRIIb extracellular region. The orientation andposition of each search model in the crystal lattice were determined inthe order of Fc CH3 domain, FcγRIIb extracellular region, and Fc CH2domain, based on the rotation function and translation function toobtain the initial model for the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex. When rigid body refinementwhich moves the two Fc CH2 domains, the two Fc CH3 domains, and theFcγRIIb extracellular region was performed on the obtained initialmodel, the crystallographic reliability factor, R value became 40.4%,and the Free R value became 41.9% to diffraction intensity data from 25Å to 3.0 Å at this point. Furthermore, structural refinement using theprogram Refmac5 (CCP4 Software Suite), and revision of the model toobserve the electron density maps whose coefficient have 2Fo-Fc orFo-Fc, which are calculated based on the experimentally determinedstructural factor Fo, the calculated structural factor Fc and thecalculated phase using the model, was carried out by the Coot program(Paul Emsley). Model refinement was carried out by repeating thesesteps. Finally, as a result of incorporation of water molecules into themodel based on the electron density maps which use 2Fo-Fc or Fo-Fc asthe coefficient, and the following refinement, the crystallographicreliability factor, R values and the Free R value of the modelcontaining 4846 non-hydrogen atoms became 23.7% and 27.6% to 24291diffraction intensity data from 25 Å to 2.6 Å resolution, respectively.

[Production of a Model Structure of the Fc (WT)/FcγRIIb ExtracellularRegion Complex]

Based on the structural coordinates of PDB code: 3RY6 which is a crystalstructure of the Fc (WT)/FcγRIIa extracellular region complex, the BuildMutants function of the Discovery Studio 3.1 program (Accelrys) was usedto introduce mutations to match the amino acid sequence of FcγRIIb intoFcγRIIa in this structural coordinates. In that case, the OptimizationLevel was set to High, Cut Radius was set to 4.5, five models weregenerated, and the one with the best energy score from among them wasset as the model structure for the Fc (WT)/FcγRIIb extracellular regioncomplex.

Reference Example 16 Analysis of FcγR Binding of Fc Variants WhoseAlteration Sites were Determined Based on Crystal Structures

Based on the results of X-ray crystal structure analysis on the complexformed between Fc (P238D) and the FcγRIIb extracellular region obtainedin Reference Example 15, variants were constructed by comprehensivelyintroducing alterations into sites on the altered Fc having substitutionof Pro at position 238 (EU numbering) with Asp that were predicted toaffect interaction with FcγRIIb (residues of positions 233, 240, 241,263, 265, 266, 267, 268, 271, 273, 295, 296, 298, 300, 323, 325, 326,327, 328, 330, 332, and 334 (EU numbering)), and whether combinations ofalterations that further enhance FcγRIIb binding in addition to theP238D alteration can be obtained, was examined.

IL6R-B3 (SEQ ID NO: 164) was produced by introducing into IL6R-G1d (SEQID NO: 156), the alteration produced by substituting Lys at position 439(EU numbering) with Glu. Next, IL6R-BF648 was produced by introducinginto IL6R-B3, the alteration produced by substituting Pro at position238 (EU numbering) with Asp. IL6R-L (SEQ ID NO: 155) was utilized as thecommon antibody L chain. These antibody variants expressed were purifiedaccording to the method of Reference Example 1. The binding of theseantibody variants to each of the FcγRs (FcγRIa, FcγRIIa type H, FcγRIIatype R, FcγRIIb, and FcγRIIIa type V) was comprehensively evaluated bythe method of Reference Example 2.

A figure was produced according to the following method to show theresults of analyzing the interactions with the respective FcγRs. Thevalue for the amount of binding of each variant to each FcγR was dividedby the value for the amount of binding of the pre-altered controlantibody (IL6R-BF648/IL6R-L, alteration by substituting Pro at position238 (EU numbering) with Asp) to each FcγR, and the obtained was thenmultiplied by 100 and shown as the relative binding activity value ofeach variant to each FcγR. The horizontal axis shows the relativebinding activity value of each variant to FcγRIIb, and the vertical axisshows the relative binding activity value of each variant to FcγRIIatype R (FIG. 40).

As shown in FIG. 40, the results show that of all the alterations, 24types of alterations were found to maintain or enhance FcγRIIb bindingin comparison with the pre-altered antibody. The binding of thesevariants to each of the FcγRs are shown in Table 27. In the table,alteration refers to the alteration introduced into IL6R-B3 (SEQ ID NO:164). The template used for producing IL6R-B3, IL6R-G1d/IL6R-L, isindicated with an asterisk (*).

TABLE 27 RELATIVE BINDING VARIANT NAME ALTERATION FcγRIa FcγRIIaRFcγRIIaH FcγRIIb FcγRIIIa IL6R-G1d/IL6R-L * 140 650 1670 62 3348IL6R-2B999/IL6R-L 145 625 1601 58 3264 IL6R-BF648/IL6R-L P238D 100 100100 100 100 IL6R-2B002/IL6R-L P238D/E233D 118 103 147 116 147IL6R-BP100/IL6R-L P238D/S267A 121 197 128 110 138 IL6R-BP102/IL6R-LP238D/S267Q 104 165 66 106 86 IL6R-BP103/IL6R-L P238D/S267V 56 163 69107 77 IL6R-BP106/IL6R-L P238D/H268D 127 150 110 116 127IL6R-BP107/IL6R-L P238D/H268E 123 147 114 118 129 IL6R-BP110/IL6R-LP238D/H268N 105 128 127 101 127 IL6R-BP112/IL6R-L P238D/P271G 119 340113 157 102 IL6R-2B128/IL6R-L P238D/Y296D 95 87 37 103 96IL6R-2B169/IL6R-L P238D/V323I 73 92 83 104 94 IL6R-2B171/IL6R-LP238D/V323L 116 117 115 113 122 IL6R-2B172/IL6R-L P238D/V323M 140 244179 132 144 IL6R-BP136/IL6R-L P238D/K326A 117 159 103 119 102IL6R-BP117/IL6R-L P238D/K326D 124 166 96 118 105 IL6R-BP120/IL6R-LP238D/K326E 125 175 92 114 103 IL6R-BP126/IL6R-L P238D/K326L 113 167 132103 146 IL6R-BP119/IL6R-L P238D/K326M 117 181 133 110 145IL6R-BP142/IL6R-L P238D/K326N 98 103 97 106 102 IL6R-BP121/IL6R-LP238D/K326Q 118 155 135 113 157 IL6R-BP118/IL6R-L P238D/K326S 101 132128 104 144 IL6R-BP116/IL6R-L P238D/K326T 110 126 110 108 114IL6R-BP911/IL6R-L P238D/A330K 52 101 108 119 120 IL6R-BP078/IL6R-LP238D/A330M 106 101 89 105 91 IL6R-BP912/IL6R-L P238D/A330R 60 81 93 10397

The results of measuring KD values of the variants shown in Table 27 forFcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIa V by the method ofReference Example 2 are summarized in Table 28. In the table, alterationrefers to the alteration introduced into IL6R-B3 (SEQ ID NO: 164). Thetemplate used for producing IL6R-B3, IL6R-G1d/IL6R-L, is indicated withan asterisk (*). Furthermore, KD (IIaR)/KD (IIb) and KD (IIaH)/KD (IIb)in the table respectively represent the value obtained by dividing theKD value of each variant for FcγRIIaR by the KD value of each variantfor FcγRIIb, and the value obtained by dividing the KD value of eachvariant for FcγRIIaH by the KD value of each variant for FcγRIIb. KD(IIb) of the parent polypeptide/KD (IIb) of the altered polypeptiderefers to the value obtained by dividing the KD value of the parentpolypeptide for FcγRIIb by the KD value of each variant for FcγRIIb. Inaddition, the KD value for the stronger of the FcγRIIaR- andFcγRIIaH-binding activities of each variant/KD value for the stronger ofthe FcγRIIaR- and FcγRIIaH-binding activities of the parent polypeptideare shown in Table 28. Here, parent polypeptide refers to the variantwhich has IL6R-B3 (SEQ ID NO: 164) as the H chain. It was determinedthat due to weak binding of FcγR to IgG, it was impossible to accuratelyanalyze by kinetic analysis, and thus the gray-filled cells in Table 28show values calculated by using Equation 2 of Reference Example 2.

KD=(R _(max)/(R _(eq)−RI)−C  [Equation 2]

TABLE 28

Table 28 shows that in comparison with IL6R-B3, all variants showedimprovement of affinity for FcγRIIb, and the range of improvement was2.1 fold to 9.7 fold. The ratio of KD value of each variant forFcγRIIaR/KD value of each variant for FcγRIIb, and the ratio of KD valueof each variant for FcγRIIaH/KD value of each variant for FcγRIIbrepresent an FcγRIIb-binding activity relative to the FcγRIIaR-bindingactivity and FcγRIIaH-binding activity, respectively. That is, thesevalues show the degree of binding selectivity of each variant forFcγRIIb, and a greater value indicates a higher binding selectivity forFcγRIIb. Since the ratio of KD value for FcγRIIaR/KD value for FcγRIIb,and the ratio of KD value for FcγRIIaH/KD value for FcγRIIb in theparent polypeptide IL6R-B3/IL6R-L were 0.3 and 0.2, respectively, allvariants in Table 28 showed improvement of binding selectivity forFcγRIIb in comparison with the parent polypeptide. When the KD value forthe stronger of the FcγRIIaR- and FcγRIIaH-binding activities of avariant/KD value for the stronger of the FcγRIIaR- and FcγRIIaH-bindingactivities of the parent polypeptide is 1 or more, this means that thestronger of the FcγRIIaR- and FcγRIIaH-binding activities of a varianthas equivalent or decreased binding compared with the binding by thestronger of the FcγRIIaR- and FcγRIIaH-binding activities of the parentpolypeptide. Since this value was 4.6 to 34.0 for the variants obtainedthis time, one may say that in comparison with the parent polypeptide,the variants obtained this time had reduced binding by the stronger ofthe FcγRIIaR- and FcγRIIaH-binding activities. These results showed thatcompared with the parent polypeptide, the variants obtained this timehave maintained or decreased FcγRIIa type R- and type H-bindingactivities, enhanced FcγRIIb-binding activity, and improved selectivityfor FcγRIIb. Furthermore, compared with IL6R-B3, all variants had loweraffinity to FcγRIa and FcγRIIIaV.

With regard to the promising variants among the obtained combinationvariants, the factors leading to their effects were studied using thecrystal structure. FIG. 41 shows the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex. In this figure, the Hchain positioned on the left side is Fc Chain A, and the H chainpositioned on the right side is Fc Chain B. Here, one can see that thesite at position 233 (EU numbering) in Fc Chain A is located near Lys atposition 113 of FcγRIIb. However, in this crystal structure, the E233side chain is in a condition of considerably high mobility, and itselectron density is not well observed. Therefore, the alterationproduced by substituting Glu at position 233 (EU numbering) with Aspleads to decrease in the degree of freedom of the side chain since theside chain becomes one carbon shorter. As a result, the entropy losswhen forming an interaction with Lys at position 113 of FcγRIIb may bedecreased, and consequently this is speculated to contribute toimprovement of binding free energy.

Similarly, FIG. 42 shows the environment near the site at position 330(EU numbering) in the structure of the Fc (P238D)/FcγRIIb extracellularregion complex. This figure shows that the environment around the siteat position 330 (EU numbering) of Fc Chain A of Fc (P238D) is ahydrophilic environment composed of Ser at position 85, Glu at position86, Lys at position 163, and such of FcγRIIb. Therefore, the alterationproduced by substituting Ala at position 330 (EU numbering) with Lys orArg is speculated to contribute to strengthening the interaction withSer at position 85 or Glu at position 86 in FcγRIIb.

FIG. 43 depicts the structures of Pro at position 271 (EU numbering) ofFc Chain B after superimposing the crystal structures of the Fc(P238D)/FcγRIIb extracellular region complex and the Fc (WT)/FcγRIIIaextracellular region complex by the least squares fitting based on theCα atom pair distances with respect to Fc Chain B. These two structuresmatch well, but have different three-dimensional structures of Pro atposition 271 (EU numbering). When the weak electron density around thisarea in the crystal structure of the Fc (P238D)/FcγRIIb extracellularregion complex is also taken into consideration, it is suggested thatthere is possibility that Pro at position 271 (EU numbering) in Fc(P238D)/FcγRIIb causes a large strain on the structure, thus disturbingthe loop structure to attain an optimal structure. Therefore, thealteration produced by substituting Pro at position 271 (EU numbering)with Gly is speculated to give flexibility to this loop structure, andcontribute to enhancement of binding by reducing the energetic barrierfor attaining an optimum structure when interacting with FcγRIIb.

Reference Example 17 Examination of the Combinatorial Effect ofAlterations that Enhance FcγRIIb Binding when Combined with P238D

Of the alterations obtained in Reference Examples 14 and 16, those thatshowed effects of enhancing FcγRIIb binding or maintaining FcγRIIbbinding and suppressing binding to other FcγRs were combined with eachother, and its effect was examined.

Particularly good alterations selected from Tables 24 and 28 wereintroduced into the antibody H chain IL6R-BF648 in a similar manner tothe method of Reference Example 16. IL6R-L was utilized as the antibodyL chain, and the expressed antibodies were purified according to themethod of Reference Example 1. The binding to each of the FcγRs (FcγRIa,FcγRIIa H type, FcγRIIa R type, FcγRIIb, and FcγRIIIa V type) wascomprehensively evaluated by the method of Reference Example 2.

According to the following method, relative binding activities werecalculated for the results of analyzing interactions with the respectiveFcγRs. The value for the amount of binding of each variant to each FcγRwas divided by the value for the amount of binding of the pre-alteredcontrol antibody (IL6R-BF648/IL6R-L with substitution of Pro at position238 (EU numbering) with Asp) to each FcγR, and multiplied by 100; andthen the value was shown as the relative binding activity value of eachvariant to each FcγR (Tables 29-1 to 29-2). In the table, alterationrefers to the alteration introduced into IL6R-B3 (SEQ ID NO: 164). Thetemplate used for producing IL6R-B3, IL6R-G1d/IL6R-L, is indicated withan asterisk (*).

TABLE 29-1 RELATIVE BINDING ACTIVITY VARIANT NAME ALTERATION FcgRIaFcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaV IL6R-G1d/IL6R-L * 140 650 1670 623348 IL6R-B3/IL6R-L 145 625 1601 58 3264 IL6R-BF648/IL6R-L P238D 100 100100 100 100 IL6R-2B253/IL6R-L E233D/P238D/V323M 155 288 207 156 126IL6R-2B261/IL6R-L E233D/P238D/Y296D 100 94 91 115 87 IL6R-BP082/ILR-LE233D/P238D/A330K 74 126 106 136 87 IL6R-BP083/IL6R-L P238D/Y296D/A330K50 87 91 122 107 IL6R-BP084/IL6R-L P238D/V323M/A330K 109 203 162 141 106IL6R-BP085/IL6R-L G237D/P238D/A330K 19 279 158 152 104 IL6R-BP086/IL6R-LP238D/K326A/A330K 72 155 116 137 123 IL6R-BP087/IL6R-L L234Y/P238D/A330K33 163 179 137 158 IL6R-BP088/IL6R-L G237D/P238D/K326A/ 25 377 166 161122 A330K IL6R-BP089/IL6R-L L234Y/P238D/K326A/ 43 222 186 147 136 A330KIL6R-BP129/IL6R-L E233D/P238D/Y296D/ 68 111 98 138 95 A330KIL6R-BP130/IL6R-L E233D/P238D/V323M/ 104 272 224 160 115 A330KIL6R-BP131/IL6R-L E233D/G237D/P238D/ 33 364 253 160 118 A330KIL6R-BP132/IL6R-L E233D/P238D/K326A/ 91 191 130 150 120 A330KIL6R-BP133/IL6R-L E233D/L234Y/P238D/ 41 174 151 137 114 A330KIL6R-BP143/IL6R-L L234Y/P238D/K326A 86 238 143 133 114 IL6R-BP144/IL6R-LG237D/P238D/K326A 64 204 108 121 128 IL6R-BP145/IL6R-L L234Y/G237D/P238D41 350 224 152 153 IL6R-BP146/IL6R-L L234Y/G237D/P238D/ 50 445 203 156180 K326A IL6R-BP147/IL6R-L L234Y/G237D/P238D/ 24 650 582 177 209K326A/A330K IL6R-BP148/IL6R-L E233D/L234Y/G237D/ 33 603 462 176 227P238D/K326A/A330K IL6R-BP149/IL6R-L E233D/L234Y/G237D/ 29 539 401 173186 P238D/Y296D/K326A/ A330K IL6R-BP150/IL6R-L L234Y/G237D/P238D/ 30 757770 183 204 K326A/A330R IL6R-BP151/IL6R-L E233D/L234Y/G237D/ 39 705 621180 221 P238D/K326A/A330R IL6R-BP152/IL6R-L E233D/L234Y/G237D/ 34 638548 178 146 P238D/Y296D/K326A/ A330R IL6R-BP176/IL6R-LE233D/P236D/K326D/ 102 201 128 147 131 A330K IL6R-BP177/IL6R-LE233D/L234Y/G237D/ 57 691 409 177 186 P238D/P271G/K326D/ A330KIL6R-BP178/IL6R-L E233D/G237D/P238D/ 51 653 259 179 110 P271G/A330KIL6R-BP179/IL6R-L G237D/P238D/P271G/ 39 570 226 177 125 K326A/A330KIL6R-BP180/IL6R-L G237D/P238D/P271G/ 29 602 203 179 100 A330K

Table 29-2 is a continuation table of Table 29-1.

TABLE 29-2 IL6R-BP181/IL6R-L E233D/P238D/P271G/ 108 362 150 170 122K326A/A330K IL6R-BP182/IL6R-L E233D/P238D/P271G/ 95 413 139 173 120Y296D/A330K IL6R-BP183/IL6R-L E233D/L234Y/P238D/ 83 423 191 164 113P271G/K326A/A330K IL6R-BP184/IL6R-L E233D/P238D/P271G/ 96 436 131 171106 A330K IL6R-BP185/IL6R-L E233D/L234Y/G237D/ 47 670 446 179 191P238D/P271G/K326A/ A330K IL6R-BP186/IL6R-L E233D/L234Y/G237D/ 43 614 368175 143 P238D/P271G/Y296D/ K326A/A330K IL6R-BP187/IL6R-LL234Y/P238D/P271G/ 68 387 205 157 124 K326A/A330K IL6R-BP188/IL6R-LE233D/G237D/P238D/ 74 636 234 179 121 H268D/P271G/A330KIL6R-BP189/IL6R-L G237D/P238D/H268D/ 56 557 183 177 141P271G/K326A/A330K IL6R-BP190/IL6R-L G237D/P238D/H268D/ 50 615 224 181155 P271G/A330K IL6R-BP191/IL6R-L E233D/P238D/H268D/ 125 382 145 170 142P271G/K326A/A330K IL6R-BP192/IL6R-L E233D/P238D/H268D/ 109 406 122 172118 P271G/Y296D/A330K IL6R-BP193/IL6R-L E233D/P238D/H268D/ 113 449 154173 135 P271G/A330K IL6R-BP194/IL6R-L E233D/L234Y/G237D/ 69 672 395 178249 P238D/H268D/P271G/ K326A/A330K IL6R-BP195/IL6R-L E233D/L234Y/G237D/68 661 344 181 221 P238D/H268D/P271G/ Y296D/K326A/A330KIL6R-BP196/IL6R-L L234Y/P238D/H268D/ 89 402 195 157 137P271G/K326A/A330K IL6R-BP197/IL6R-L E233D/L234Y/G237D/ 71 642 294 179206 P238D/H268D/P271G/ Y296D/K326D/A330K IL6R-BP198/IL6R-LE233D/L234Y/P238D/ 104 449 188 164 157 H268D/P271G/K326A/ A330KIL6R-BP199/IL6R-L E233D/P238D/K326D/ 112 172 116 144 103 A330RIL6R-BP200/IL6R-L E233D/L234Y/G237D/ 60 754 517 188 164P238D/P271G/K326D/ A330R IL6R-BP201/IL6R-L E233D/G237D/P238D/ 57 696 359186 121 P271G/A330R IL6R-BP202/IL6R-L G237D/P238D/P271G/ 43 615 285 185108 K326A/A330R IL6R-BP203/IL6R-L G237D/P238D/P271G/ 35 637 255 185 88A330R IL6R-BP204/IL6R-L E233D/P238D/P271G/ 110 301 137 165 121K326A/A330R IL6R-BP205/IL6R-L E233D/P238D/P271G/ 97 335 108 167 93Y296D/A330R IL6R-BP206/IL6R-L E233D/P238D/P271G/ 101 362 123 168 92A330R IL6R-BP207/IL6R-L E233D/P238D/A330R 74 103 103 124 97IL6R-BP208/IL6R-L E233D/G237D/P238D/ 81 690 310 188 118H268D/P271G/A330R IL6R-BP209/IL6R-L G237D/P238D/H268D/ 68 625 267 186153 P271G/K326A/A330R IL6R-BP210/IL6R-L G237D/P238D/H268D/ 57 661 279187 135 P271G/A330R IL6R-BP211/IL6R-L E233D/P238D/H268D/ 128 312 111 16587 P271G/K326A/A330R IL6R-BP212/IL6R-L E233D/P238D/H268D/ 117 363 135173 122 P271G/Y296D/A330R IL6R-BP213/IL6R-L E233D/P238D/H268D/ 118 382123 169 100 P271G/A330R IL6R-BP214/IL6R-L E233D/L234Y/G237D/ 36 498 285174 165 P238D/Y296D/K326D/ A330K

The results of measuring KD values of the variants shown in Tables 29-1and 29-2 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIaV by themethod of Reference Example 2 are summarized in Tables 30-1 and 30-2. Inthe table, alteration refers to the alteration introduced into IL6R-B3(SEQ ID NO: 164). The template used for producing IL6R-B3,IL6R-G1d/IL6R-L, is indicated with an asterisk (*). Furthermore, KD(IIaR)/KD (IIb) and KD (IIaH)/KD (IIb) in the table respectivelyrepresent the value obtained by dividing the KD value of the variant forFcγRIIaR by the KD value of the variant for FcγRIIb, and the valueobtained by dividing the KD value of the variant for FcγRIIaH by the KDvalue of each variant for FcγRIIb. KD (IIb) of the parent polypeptide/KD(IIb) of the altered polypeptide refers to the value obtained bydividing the KD value of the parent polypeptide for FcγRIIb by the KDvalue of each variant for FcγRIIb. In addition, the KD value for thestronger of the FcγRIIaR- and FcγRIIaH-binding activities of eachvariant/KD value for the stronger of the FcγRIIaR- and FcγRIIaH-bindingactivities of the parent polypeptide are shown in Tables 30-1 and 30-2.Here, parent polypeptide refers to the variant which has IL6R-B3 (SEQ IDNO: 164) as the H chain. It was determined that due to weak binding ofFcγR to IgG, it was impossible to accurately analyze by kineticanalysis, and thus the values of gray-filled cells in Tables 30-1 and30-2 show values calculated by using Equation 2 of Reference Example 2.

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

Tables 30-1 and 30-2 show that in comparison with IL6R-B3, all variantsshowed improvement of affinity for FcγRIIb, and the range of improvementwas 3.0 fold to 99.0 fold. The ratio of KD value of each variant forFcγRIIaR/KD value of each variant for FcγRIIb, and the ratio of KD valueof each variant for FcγRIIaH/KD value of each variant for FcγRIIbrepresent an FcγRIIb-binding activity relative to the FcγRIIaR-bindingactivity and FcγRIIaH-binding activity, respectively. That is, thosevalues show the degree of binding selectivity of each variant forFcγRIIb, and a greater value indicates a higher binding selectivity forFcγRIIb. Since the ratio of KD value for FcγRIIaR/KD value for FcγRIIb,and the ratio of KD value for FcγRIIaH/KD value for FcγRIIb of theparent polypeptide IL6R-B3/IL6R-L were 0.3 and 0.2, respectively, allvariants in Tables 30-1 and 30-2 showed improvement of bindingselectivity for FcγRIIb in comparison with the parent polypeptide. Whenthe KD value for the stronger of the FcγRIIaR- and FcγRIIaH-bindingactivities of a variant/KD value for the stronger of the FcγRIIaR- andFcγRIIaH-binding activities of the parent polypeptide is 1 or more, thismeans that the stronger of the FcγRIIaR- and FcγRIIaH-binding activitiesof a variant has equivalent or decreased binding compared with thebinding by the stronger of the FcγRIIaR- and FcγRIIaH-binding activitiesof the parent polypeptide. Since this value was 0.7 to 29.9 for thevariants obtained this time, one may say that binding by the stronger ofthe FcγRIIaR- and FcγRIIaH-binding activities of the variants obtainedthis time was nearly equivalent or decreased compared with that of theparent polypeptide. These results showed that compared with the parentpolypeptide, the variants obtained this time have maintained ordecreased FcγRIIa type R— and type H-binding activities, enhancedFcγRIIb-binding activity, and improved selectivity for FcγRIIb.Furthermore, compared with IL6R-B3, all variants had lower affinity forFcγRIa and FcγRIIIaV.

TABLE 30-1

Table 30-2 is a continuation table of Table 30-1.

TABLE 30-2

Reference Example 18 Preparation of Variants with Enhanced FcγRIIbBinding

As shown in Reference Example 13, when enhancing the FcγRIIb binding, itis preferable that the FcγRIIb binding is enhanced while maximallysuppressing the binding to other activating FcγRs. Thus, the presentinventors additionally produced variants with enhanced FcγRIIb bindingor improved selectivity to FcγRIIb by combining alterations that enhancethe FcγRIIb binding or improve the selectivity to FcγRIIb. Specifically,the alterations described in Reference Examples 14, 16, and 17 whichwere found to be effective when combined with alteration P238D, werecombined with one another, on the basis of the P238D alteration whichshowed the excellent effect to enhance the FcγRIIb binding and toimprove the selectivity to FcγRIIb.

Variants were produced by combining the Fc regions of IL6R-G1d (SEQ IDNO: 156) and IL6R-B3 (SEQ ID NO: 164) with alterations E233D, L234Y,G237D, S267Q, H268D, P271G, Y296D, K326D, K326A, A330R, and A330Kdescribed in Reference Examples 14, 16, and 17 which were found to beeffective when combined with alteration P238D. Using IL6R-L (SEQ ID NO:155) as the antibody L chain, antibodies comprising the above-describedvariants in the heavy chain were expressed and purified according to themethod described in Reference Example 1. The resulting variants wererespectively assessed for the binding to each FcγR (FcγRIa, FcγRIIaH,FcγRIIaR, FcγRIIb, or FcγRIIIaV) by the method described in ReferenceExample 2.

The KD value of each variant to each FcγR is shown in Table 31.“Alteration” refers to an alteration introduced into IL6R-B3 (SEQ ID NO:164). IL6R-B3/IL6R-L which is used as the template to produce eachvariant is indicated by asterisk (*). KD (IIaR)/KD (IIb) in the tableshows the value obtained by dividing the KD value of each variant forFcγRIIaR by the KD value of each variant for FcγRIIb. The greater thevalue, the higher the selectivity to FcγRIIb. KD (IIb) of parentpolypeptide/KD (IIb) of altered polypeptide shows the value obtained bydividing the KD value of IL6R-B3/IL6R-L for FcγRIIb by the KD value ofeach variant for FcγRIIb. Meanwhile, KD (IIaR) of parent polypeptide/KD(IIaR) of altered polypeptide shows the value obtained by dividing theKD value of IL6R-B3/IL6R-L for FcγRIIaR by the KD value of each variantfor FcγRIIaR. In Table 31, the numeral in the gray-filled cellsindicates that the binding of FcγR to IgG was concluded to be too weakto analyze correctly by kinetic analysis and thus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

TABLE 31

When taking the binding to each FcγR by IL6R-B3/IL6R-L resulting fromintroducing the K439E alteration into IL6R-G1d/IL6R-L containing thesequence of native human IgG1 as 1, the binding of IL6R-G1d/IL6R-L toFcγRIa was 1.3 times; the binding of IL6R-G1d/IL6R-L to FcγRIIaR was 1.1times; the binding of IL6R-G1d/IL6R-L to FcγRIIaH was 1.1 times, thebinding of IL6R-G1d/IL6R-L to FcγRIIb was 1.2 times, and the binding ofIL6R-G1d/IL6R-L to FcγRIIIaV was 0.9 times. Thus, for any given FcγRtype, the binding of IL6R-B3/IL6R-L to FcγR was comparable to thebinding of IL6R-G1d/IL6R-L to FcγR. Thus, the comparison of the bindingof each variant to each FcγR with that of IL6R-B3/IL6R-L prior tointroduction of the alteration is assumed to be equivalent to thecomparison of the binding of each variant to each FcγR with the bindingto each FcγR by IL6R-G1d/IL6R-L containing the sequence of native humanIgG1. For this reason, in the subsequent Examples below, the bindingactivity of each variant to each FcγR will be compared to the binding toeach FcγR by IL6R-B3/IL6R-L prior to introduction of the alteration.Table 31 shows that all the variants have increased FcγRIIb bindingactivity as compared to IL6R-B3 prior to introduction of the alteration.The binding activity of IL6R-BF648/IL6R-L, which was the lowest, wasincreased by 2.6 times, while the binding activity of IL6R-BP230/IL6R-L,which was the highest, was increased by 147.6 times. Regarding the valueof KD (IIaR)/KD (IIb) that represents the selectivity, the value forIL6R-BP234/IL6R-L, which was the lowest, was 10.0, while the value forIL6R-BP231/IL6R-L, which was the highest, was 32.2. Compared to 0.3 forIL6R-B3/IL6R-L prior to introduction of the alteration, the values implythat all the variants have improved selectivity. All the variants showedlower binding activity to FcγRIa, FcγRIIaH, and FcγRIIIaV thanIL6R-B3/IL6R-L prior to introduction of the alteration.

Reference Example 19 X-Ray Crystal Structure Analysis of the Complexesof FcγRIIb Extracellular Region or FcγRIIaR Extracellular Region and FcRegion with Enhanced FcγRIIb Binding

As shown in Reference Example 18, the FcγRIIb binding of variantIL6R-BP230/IL6R-L, whose FcγRIIb binding was enhanced most, was enhancedto about 150 times as compared to IL6R-B3/IL6R-L prior to introductionof the alteration, while the enhancement of its FcγRIIaR binding wassuppressed to an extent of about 1.9 times. Thus, IL6R-BP230/IL6R-L is avariant excellent in both FcγRIIb binding and selectivity. However, thepresent inventors sought a possibility to create more preferablevariants with further enhanced FcγRIIb binding while suppressing theFcγRIIaR binding as possible.

As shown in FIG. 39 described in Reference Example 15, in the Fc regionwith alteration P238D, Asp at position 270 (EU numbering) in its CH2domain B forms a tight electrostatic interaction with Arg at position131 in FcγRIIb. This amino acid residue at position 131 is His inFcγRIIIa and FcγRIIaH, while it is Arg in FcγRIIaR like in FcγRIIb.Thus, there is no difference between FcγRIIaR and FcγRIIb in terms ofthe interaction of the amino acid residue at position 131 with Asp atposition 270 (EU numbering) in the CH2 domain B. This is assumed to be amajor factor for the poor selectivity between the FcγRIIb binding andFcγRIIaR binding of the Fc region.

On the other hand, the extracellular regions of FcγRIIa and FcγRIIb are93% identical in amino acid sequence, and thus they share very highhomology. Based on the crystal structure analysis of the complex of theFc region of native IgG1 (hereinafter abbreviated as Fc (WT)) and theextracellular region of FcγRIIaR (J. Immunol. (2011) 187, 3208-3217), adifference found around the interface of their interaction was onlythree amino acids (G1n127, Leu132, Phe160) between FcγRIIaR and FcγRIIb.Thus, the present inventors predicted that it was extremely difficult toimprove the selectivity of the Fc region between the FcγRIIb binding andFcγRIIaR binding.

In this context, the present inventors conceived that, in order tofurther enhance the FcγRIIb-binding activity of the Fc region and toimprove the selectivity of its FcγRIIaR binding, it was important toclarify subtle differences between Fc region-FcγRIIb interaction and Fcregion-FcγRIIaR interaction by analyzing not only the three-dimensionalstructure of the complex of the Fc region with enhanced FcγRIIb bindingand the extracellular region of FcγRIIb but also the three-dimensionalstructure of the complex of the Fc region with enhanced FcγRIIb bindingand the extracellular region of FcγRIIaR. First, the present inventorsanalyzed the X-ray crystal structure of the complex of the extracellularregion of FcγRIIb or FcγRIIaR and Fc (P208) resulting from eliminatingthe K439E alteration from the Fc region of IL6R-BP208/IL6R-L created asdescribed in Reference Example 17, which was the variant used as thebasis in producing IL6R-BP230/IL6R-L.

(19-1) X-Ray Crystal Structure Analysis of the Complex of Fc (P208) andthe Extracellular Region of FcγRIIb [Expression and Purification of Fc(P208)]

Fc (P208) was prepared as described below. First, IL6R-P208 was producedby substituting Lys for Glu at position 439 (EU numbering) inIL6R-BP208, as is in the case of the sequence of native human IgG1.Then, the gene sequence of Fc (P208) spanning from Glu at position 216(EU numbering) to the C terminus was cloned by PCR using as a template aDNA encoding a variant with a substitution of Ser for Cys at position220 (EU numbering). Expression vector construction, expression, andpurification were achieved according to the method described inReference Example 1. Meanwhile, Cys at position 220 (EU numbering) inordinary IgG1 forms a disulfide bond to a Cys in the L chain. Whenpreparing the Fc region alone, the L chain is not coexpressed. Thus, Cysat position 220 was substituted by Ser to avoid unnecessary disulfidebond formation.

[Expression and Purification of the Extracellular Region of FcγRIIb]

The extracellular region of FcγRIIb was prepared according to the methoddescribed in Reference Example 2.

[Purification of the Fc (P208)/FcγRIIb Extracellular Region Complex]

0.15 mg of the purified product of Endo F1 (Protein Science (1996) 5,2617-2622) expressed in E. coli as a fusion protein with glutathioneS-transferase was added 1.5 mg of a crystallization sample of theextracellular region of FcγRIIb. This added sample in 0.1 M Bis-Trisbuffer (pH 6.5) was allowed to stand at room temperature for three daysto cleave off N-type sugar chains except N-acetylglucosamine directlylinked to the Asn in the sample of the extracellular region of FcγRIIb.Then, the sample of the extracellular region of FcγRIIb subjected to thesugar chain cleavage treatment was concentrated with a 5000MWCOultrafiltration filter, and purified by chromatography with a gelfiltration column (Superdex200 10/300) equilibrated with 20 mM HEPES(pH7.5)/0.1 M NaCl. Next, Fc (P208) was added in such a way that theextracellular region of FcγRIIb is present in a slightly excessive molarratio. After concentrating with a 10000MWCO ultrafiltration filter, thepurified fraction of the extracellular region of FcγRIIb subjected tothe sugar chain cleavage was purified by chromatography with a gelfiltration column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH7.5)/0.1 M NaCl. The purified fraction prepared as described above wasused as a sample of Fc (P208)/FcγRIIb extracellular region complex inthe subsequent assessment.

[Crystallization of the Complex of Fc (P208)/FcγRIIb ExtracellularRegion]

A sample of Fc (P208)/FcγRIIb extracellular region complex concentratedto about 10 mg/ml with a 10000MWCO ultrafiltration filter wascrystallized using the hanging drop vapor diffusion method incombination with the seeding method. VDXm plate (Hampton Research) wasused for crystallization. Using a reservoir solution of 0.1 M Bis-Tris(pH 6.5)/19% (w/v) PEG3350/0.2 M potassium phosphate dibasic,crystallization drops were prepared at a mixing ratio of reservoirsolution:crystallization sample=0.85 μl:0.85 μl. Crystals of the complexobtained under the same condition were crushed with Seed Bead (HamptonResearch) to prepare a seed crystal solution. The crystallization dropswere added with 0.15 μl of a diluted solution prepared from the seedsolution and allowed to stand at 20° C. in sealed reservoir wells. Thisyielded plate-like crystals.

[X-Ray Diffraction Data Measurements from an Fc (P208)/FcγRIIbExtracellular Region Complex Crystal]

A single crystal of Fc (P208)/FcγRIIb extracellular region complexprepared as described above was soaked into a solution of 0.1 M Bis-Tris(pH 6.5)/24% (w/v) PEG3350/0.2 M potassium phosphate dibasic/20% (v/v)ethylene glycol. Then, the single crystal was fished out of the solutionusing a pin with attached tiny nylon loop, and frozen in liquidnitrogen. X-ray diffraction data of the single crystal was collectedwith Spring-8 BL32XU. During the measurement, the crystal was constantlyplaced in a nitrogen stream at −178° C. to maintain in a frozen state. Atotal of 300 X-ray diffraction images of the single crystal werecollected using CCD detector MX-225HE (RAYONIX) attached to a beam linewith rotating the single crystal 0.6° at a time. Based on the obtaineddiffraction images, lattice constant determination, diffraction spotindexing, and diffraction data processing were performed using programsXia2 (J. Appl. Cryst. (2010) 43, 186-190), XDS Package (Acta Cryst.(2010) D66, 125-132) and Scala (Acta Cryst. (2006) D62, 72-82). Finally,diffraction intensity data up to 2.81 Å resolution was obtained. Thecrystal belongs to the space group C222₁ with lattice constant a=156.69Å, b=260.17 Å, c=56.85 Å, α=90°, β=90°, and γ=90°.

[X-Ray Crystal Structure Analysis of Fc (P208)/FcγRIIb ExtracellularRegion Complex]

The structure of Fc (P208)/FcγRIIb extracellular region complex wasdetermined by a molecular replacement method using program Phaser (J.Appl. Cryst. (2007) 40, 658-674). The number of complexes in anasymmetrical unit was estimated to be one from the size of the obtainedcrystal lattice and the molecular weight of Fc (P208)/FcγRIIbextracellular region complex. The segments spanning the amino acidresidues at positions 239-340 of the A chain and at positions 239-340 ofthe B chain, which were retrieved as an independent coordinate from thestructural coordinate of PDB code: 3SGJ for the crystal structure of Fc(WT)/FcγRIIIa extracellular region complex, were used as a model forsearching the CH2 domain of the Fc region. Likewise, the segmentsspanning the amino acid residues at positions 341-444 of the A chain andat positions 341-443 of the B chain, which were retrieved as acoordinate from the structural coordinate of PDB code: 3SGJ, were usedas a model for searching the CH3 domain of the Fc region. Finally, thesegment spanning the amino acid residues at positions 6-178 of the Achain, which was retrieved from the structural coordinate of PDB code:2FCB for the crystal structure of the extracellular region of FcγRIIb,was used as a model for searching Fc (P208). The present inventors triedto determine the orientations and positions of the respective searchmodels of the CH3 domain of the Fc region, the extracellular region ofFcγRIIb, and the CH2 domain of the Fc region in the crystal latticesbased on the rotation function and translation function, but failed todetermine the position of one of the CH2 domains. Then, with referenceto the crystal structure of the complex of Fc (WT)/FcγRIIIaextracellular region, the position of the last CH2 domain A wasdetermined from an electron density map that was calculated based on thephase determined for the remaining three parts. Thus, the presentinventors obtained an initial model for the crystal structure of thecomplex of Fc (P208)/FcγRIIb extracellular region. The crystallographicreliability factor R value of the structural model for the data ofdiffracted intensity at 25 to 3.0 Å was 42.6% and Free R value was 43.7%after rigid body refinement where the two CH2 domains and two CH3domains of the Fc region, and the extracellular region of FcγRIIb wereallowed to deviate from the obtained initial structural model. Then,structural model refinement was achieved by repeating structuralrefinement using program REFMAC5 (Acta Cryst. (2011) D67, 355-367)followed by revision of the structural model performed using programCoot (Acta Cryst. (2010) D66, 486-501) with reference to the electrondensity maps where the coefficients 2Fo-Fc and Fo-Fc were calculatedusing experimentally determined structural factor Fo, structural factorFc calculated according to the structural model, and the phasescalculated according to the structural model. Then, further refinementwas carried out based on the electron density maps with coefficients of2Fo-Fc and Fo-Fc by integrating water molecules into the structuralmodel. With 27259 diffracted intensity data at 25 to 2.81 Å resolution,ultimately the crystallographic reliability factor R value was 24.5% andfree R value was 28.2% for the structural model comprising 4786non-hydrogen atoms.

The three-dimensional structure of the complex of Fc (P208)/FcγRIIbextracellular region was determined at a resolution of 2.81 Å bystructure analysis. The structure obtained by the analysis is shown inFIG. 44. FcγRIIb extracellular region was revealed to be bound andsandwiched between the two CH2 domains of the Fc region, which resemblesthe three-dimensional structures of the previously analyzed complexes ofFc (WT), which is the Fc of native IgG, and each of the extracellularregions of FcγRIIIa (Proc. Natl. Acad. Sci. USA (2011) 108,12669-126674), FcγRIIIb (Nature (2000) 400, 267-273; J. Biol. Chem.(2011) 276, 16469-16477), and FcγRIIa (J. Immunol. (2011) 187 (6),3208-3217).

A close observation of the complex of Fc (P208)/FcγRIIb extracellularregion revealed a change in the loop structure at positions 233 to 239(EU numbering) following the hinge region in the CH2 domain A of the Fcregion due to an influence of the introduced the G237D and P238Dalterations as compared to the complex of Fc (WT)/FcγRIIaR extracellularregion (FIG. 45). This leads to that the main chain of Asp at position237 (EU numbering) in Fc (P208) formed a tight hydrogen bond to the sidechain of Tyr at position 160 in FcγRIIb (FIG. 46). In both FcγRIIaH andFcγRIIaR, the amino acid residue at position 160 is Phe, which isincapable of forming such a hydrogen bond. This suggests that the abovedescribed hydrogen bond has important contribution to the enhancement ofthe FcγRIIb binding and the acquisition of the FcγRIIa bindingselectivity of Fc (P208), i.e., improvement of the FcγRIIb-bindingactivity and reduction of FcγRIIa-binding activity of Fc (P208).

On the other hand, the side chain of Asp at position 237 (EU numbering)in Fc (P208) forms neither particularly significant interaction in theFcγRIIb binding nor interaction with other residues within the Fcregion. Ile at position 332, Glu at position 333, and Lys at position334 (EU numbering) in the Fc region are located close to Asp at position237 (EU numbering) (FIG. 47). When the amino acid residues of thesepositions are substituted by hydrophilic residues to form an interactionwith the side chain of Asp at position 237 (EU numbering) in Fc (P208)and the loop structure can be stabilized by the interaction, this canlead to reduction of the entropic energy loss due to the hydrogenbonding between the Fc region and Tyr at position 160 in FcγRIIb andthereby to an increase in the binding free energy, i.e., an increase inthe binding activity.

When the X-ray crystal structure of the complex of Fc (P238D) with theP238D alteration and FcγRIIb extracellular region described in ReferenceExample 15 is compared to the X-ray crystal structure of the complex ofFc (P208) and FcγRIIb extracellular region, deviations are observed atfive portions in Fc (P208) as compared to Fc (P238D) and most of thechanges are seen only at the side chain level. Meanwhile, a positionaldeviation at the main chain level due to the Pro-to-Gly alteration atposition 271 (EU numbering) is also observed in the CH2 domain B of theFc region, and in addition there is a structural change in the loop atpositions 266 to 270 (EU numbering) (FIG. 48). As described in ReferenceExample 16, it is suggested that, when Asp at position 270 (EUnumbering) in Fc (P238D) forms a tight electrostatic interaction withArg at position 131 in FcγRIIb, the interaction can inducestereochemical stress at Pro at position 271 (EU numbering). Theexperiment described herein suggests that the structural change observedwith the alteration to Gly for the amino acid at position 271 (EUnumbering) is assumed to be a result of elimination of the structuraldistortion accumulated at Pro prior to the alteration and theelimination results in an increase in the free energy for the FcγRIIbbinding, i.e., an increase in the binding activity.

Furthermore, it was demonstrated that, due to the change of the loopstructure at positions 266 to 271 (EU numbering), Arg at position 292(EU numbering) underwent a structural change while being in two states.In this case, the electrostatic interaction (FIG. 48) formed between Argat position 292 (EU numbering) and Asp at position 268 (EU numbering)which is an altered residue in Fc (P208) can contribute to thestabilization of the loop structure. Since the electrostatic interactionformed between Asp at position 270 (EU numbering) in the loop and Arg atposition 131 in FcγRIIb largely contribute to the binding activity of Fc(P208) to FcγRIIb, the stabilization of the loop structure in thebinding conformation was likely to reduce the entropic energy loss uponbinding. Thus, the alteration is expected to result in an increase inthe binding free energy, i.e., an increase in the binding activity.

Moreover, the possibility of alteration to further increase the activitywas scrutinized based on the result of structural analysis. Ser atposition 239 (EU numbering) was found as a candidate for the site tointroduce alteration. As shown in FIG. 49, Ser at position 239 (EUnumbering) in the CH2 domain B is present at the position toward whichLys at position 117 in FcγRIIb extends most naturally in structure.However, since the electron density was not observed for Lys at position117 in FcγRIIb by the analysis described above, the Lys has no definitestructure. In this situation, Lys117 is likely to have only a limitedeffect on the interaction with Fc (P208). When Ser at position 239 (EUnumbering) in the CH2 domain B is substituted with negatively chargedAsp or Glu, such an alteration is expected to cause an electrostaticinteraction with the positively charged Lys at position 117 in FcγRIIb,thereby resulting in improved FcγRIIb-binding activity.

On the other hand, an observation of the structure of Ser at position239 (EU numbering) in the CH2 domain A revealed that, by forming ahydrogen bond to the main chain of Gly at position 236 (EU numbering),the side chain of this Ser stabilized the loop structure at positions233 to 239, including Asp at position 237 (EU numbering) that forms ahydrogen bond to the side chain of Tyr at position 160 in FcγRIIb,following the hinge region (FIG. 46). The stabilization of the loopstructure in the binding conformation can reduce the entropic energyloss upon binding, and result in an increase in the binding free energy,i.e., an improvement of the binding activity. Meanwhile, when Ser atposition 239 (EU numbering) in the CH2 domain A is substituted with Aspor Glu, the loop structure can become unstable due to loss of thehydrogen bond to the main chain of Gly at position 236 (EU numbering).In addition, the alteration can result in electrostatic repulsion to Aspat position 265 (EU numbering) in close proximity, leading to furtherdestabilization of the loop structure. The energy for thedestabilization corresponds to loss of free energy for the FcγRIIbbinding, which can result in reduction in the binding activity.

(19-2) X-Ray Crystal Structure Analysis of the Complex of Fc (P208) andFcγRIIaR Extracellular Region [Expression and Purification of theExtracellular Region of FcγRIIaR]

The extracellular region of FcγRIIaR was prepared according to themethod described in Reference Example 2.

[Purification of the Complex of Fc (P208)/FcγRIIaR Extracellular Region]

1.5 mg of purified sample of the extracellular region of FcγRIIaR wasadded with 0.15 mg of the purified product of Endo F1 (Protein Science(1996) 5, 2617-2622) expressed in E. coli as a fusion protein withglutathione S-transferase, 20 μl of 5 U/ml Endo F2 (QA-bio), and 20 μlof 5 U/ml Endo F3 (QA-bio). After 9 days of incubation at roomtemperature in 0.1 M Na acetate buffer (pH 4.5), the sample was furtheradded with 0.07 mg of the above-described Endo F1, 7.5 μl of theabove-described Endo F2, and 7.5 μl of the above-described Endo F3, andwas incubated for three days to cleave off N-type sugar chains exceptN-acetylglucosamine directly linked to the Asn in the sample of theextracellular region of FcγRIIa R. Then, the sample of the extracellularregion of FcγRIIaR concentrated with a 10000MWCO ultrafiltration filterand subjected to the above-described sugar chain cleavage treatment waspurified by chromatography with a gel filtration column (Superdex20010/300) equilibrated with 25 mM HEPES (pH 7)/0.1M NaCl. Next, Fc (P208)was added in such a way that the extracellular region of FcγRIIaR ispresent in a slightly excessive molar ratio. After concentrating with a10000MWCO ultrafiltration filter, the purified fraction of theextracellular region of FcγRIIaR subjected to the above-described sugarchain cleavage treatment was purified by chromatography with a gelfiltration column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH7)/0.1 M NaCl. The purified fraction prepared as described above wasused as a sample of Fc (P208)/FcγRIIaR extracellular region complex inthe subsequent assessment.

[Crystallization of the Complex of Fc (P208)/FcγRIIaR ExtracellularRegion]

A sample of Fc (P208)/FcγRIIa R extracellular region complexconcentrated to about 10 mg/ml with a 10000MWCO ultrafiltration filterwas crystallized using the sitting drop vapor diffusion method. Using areservoir solution of 0.1 M Bis-Tris (pH 7.5)/26% (w/v) PEG3350/0.2 Mammonium sulfate, crystallization drops were prepared at a mixing ratioof reservoir solution:crystallization sample=0.8 μl: 1.0 μl. The dropswere tight sealed and allowed to stand at 20° C. This yielded plate-likecrystals.

[X-Ray Diffraction Data Measurement from Fc (P208)/FcγRIIaRExtracellular Region Complex Crystal]

A single crystal of Fc (P208)/FcγRIIaR extracellular region complexprepared as described above was soaked into a solution of 0.1 M Bis-Tris(pH 7.5)/27.5% (w/v) PEG3350/0.2 M ammonium sulfate/20% (v/v) glycerol.Then, the crystal was fished out of the solution using a pin withattached tiny nylon loop, and frozen in liquid nitrogen. X-raydiffraction data of the single crystal was collected from Photon FactoryBL-17A of the synchrotron radiation institution in the High EnergyAccelerator Research Organization. The crystal was constantly placed ina nitrogen stream at −178° C. to maintain in a frozen state during themeasurement. A total of 225 X-ray diffraction images of the singlecrystal were collected using CCD detector Quantum 315r (ADSC) equippedto the beam line with rotating the single crystal at 0.6° at a time.Based on the obtained diffraction images, lattice constantdetermination, diffraction spot indexing, and diffraction dataprocessing were performed using programs Xia2 (J. Appl. Cryst. (2010)43, 186-190), XDS Package (Acta Cryst. (2010) D66, 125-132), and Scala(Acta Cryst. (2006) D62, 72-82). Finally, diffraction intensity data upto 2.87 Å resolution was obtained. The crystal belongs to the spacegroup C222₁ with lattice constant a=154.31 Å, b=257.61 Å, c=56.19 Å,α=90°, β=90°, and γ=90°.

[X-Ray Crystal Structure Analysis of Fc (P208)/FcγRIIaR ExtracellularRegion Complex]

The structure of Fc (P208)/FcγRIIaR extracellular region complex wasdetermined by a molecular replacement method using program Phaser (J.Appl. Cryst. (2007) 40, 658-674). The number of complexes in anasymmetrical unit was estimated to be one from the size of the obtainedcrystal lattice and the molecular weight of Fc (P208)/FcγRIIaRextracellular region complex. Using, as a search model, thecrystallographic structure of Fc (P208)/FcγRIIb extracellular regioncomplex obtained as described in (19-1), the orientation and position ofFc (P208)/FcγRIIaR extracellular region complex in the crystal latticeswere determined based on the rotation function and translation function.The crystallographic reliability factor R value of the structural modelfor the data of diffracted intensity at 25 to 3.0 Å was 38.4% and Free Rvalue was 30.0% after rigid body refinement where the two CH2 domainsand two CH3 domains of the Fc region, and the extracellular region ofFcγRIIaR were allowed to independently deviate from the obtained initialstructural model. Then, structural model refinement was achieved byrepeating structural refinement using program REFMAC5 (Acta Cryst.(2011) D67, 355-367) followed by revision of the structural modelperformed using program Coot (Acta Cryst. (2010) D66, 486-501) withreference to the electron density maps where the coefficients Fo-Fc and2Fo-Fc were calculated using experimentally determined structural factorFo, structural factor Fc calculated according to the model, and thephases calculated according to the model. Finally, further refinementwas carried out based on the electron density maps with coefficientsFo-Fc and 2Fo-Fc by integrating water molecules into the structuralmodel. With 24838 diffracted intensity data at 25 to 2.87 Å resolution,ultimately the crystallographic reliability factor R value was 26.3% andfree R value was 38.0% for the structural model comprising 4758non-hydrogen atoms.

The three-dimensional structure of the complex of Fc (P208)/FcγRIIaRextracellular region was determined at a resolution of 2.87 Å bystructure analysis. A comparison of the crystal structure between thecomplex of Fc (P208)/FcγRIIaR extracellular region and the complex of Fc(P208)/FcγRIIb extracellular region described in (19-1) detected almostno difference at the level of overall structure (FIG. 50), reflectingthe very high amino acid identity between the two Fcγ receptors.

However, a precise observation of the structures at the electron densitylevel detected some differences that can lead to improvement of theselectivity between the FcγRIIb binding and the FcγRIIaR binding of theFc region. The amino acid residue at position 160 in FcγRIIaR is not Tyrbut Phe. As shown in FIG. 51, the hydrogen bond between the main chainof the amino acid residue at position 237 (EU numbering) in the CH2domain A of the Fc region and Tyr at position 160 in FcγRIIb, thoughformed upon binding between FcγRIIb and the Fc region with alterationP238D, is expected not to be formed upon binding between FcγRIIaR andthe Fc region with alteration P238D. The absence of the hydrogen bondformation can be a major factor for improving the selectivity betweenthe FcγRIIb binding and the FcγRIIaR binding of the Fc region introducedwith alteration P238D. Further comparison at the electron density levelshowed that, in the Fc region/FcγRIIb complex, electron density wasclearly observable for the side chains of Leu at positions 235 and 234(EU numbering), whereas the electron density of the side chains wasunclear in the Fc region/FcγRIIaR complex. This suggests that the loopnear position 237 (EU numbering) fluctuates due to the reduced FcγRIIaRinteraction around this position. Meanwhile, a structural comparison ofthe CH2 domain B of the Fc region (FIG. 52) in same region revealedthat, in the complex of the Fc region and FcγRIIb, electron density wasobservable up to Asp at position 237 (EU numbering), whereas, in thecomplex of the Fc region and FcγRIIaR, electron density was observableup to three residues prior to Asp at position 237 (EU numbering), i.e.,up to around Leu at position 234 (EU numbering), suggesting thatFcγRIIaR binding forms an interaction over a larger region as comparedto the FcγRIIb binding. The finding described above suggests thepossibility that, in the CH2 domain A of the Fc region, the region fromposition 234 to 238 (EU numbering) has a large contribution to thebinding between the Fc region and FcγRIIb, while in the CH2 domain B ofthe Fc region the region from position 234 to 238 (EU numbering) has alarge contribution to the binding between the Fc region and FcγRIIaR.

Reference Example 20 Fc Variants for which Alteration Sites wereDetermined Based on Crystal Structure

As described in Reference Example 19, Asp at position 268 (EU numbering)was suggested to electrostatically interact with Arg at position 292 (EUnumbering) (FIG. 48) as a result of the local structural change due tointroduction of the alteration P271G in domain B of the variant withenhanced FcγRIIb binding (P208). There is a possibility that the loopstructure at positions 266 to 271 (EU numbering) is stabilized by theformation of the interaction, resulting in enhancement of the FcγRIIbbinding. Thus, the present inventors assessed whether the FcγRIIbbinding of the variant could be enhanced by additional stabilization ofits loop structure due to enhancement of the electrostatic interactionby substituting Glu for Asp at position 268 (EU numbering) in thevariant. On the other hand, as shown in FIG. 47, Tyr at position 160 inFcγRIIb interacts with the main chain of Asp at position 237 (EUnumbering) in domain A of P208. Meanwhile, the side chain of Asp atposition 237 (EU numbering) is located close to Ile at position 332, Gluat position 333, and Lys at position 334 (EU numbering) in the moleculewithout forming any particularly significant interaction. Thus, thepresent inventors also assessed whether the interaction with Tyr atposition 160 in FcγRIIb can be enhanced through stabilization of theloop structure at positions 266 to 271 (EU numbering) due to increasedinteraction with the side chain of Asp at position 237 (EU numbering) bysubstituting hydrophilic amino acid residues at the positions describedabove.

Variants were produced by introducing each of alterations H268E, I332T,I332S, I332E, I332K, E333K, E333R, E333S, E333T, K334S, K334T, and K334Einto IL6R-BP230/IL6R-L produced as described in Reference Example 18.IL6R-L (SEQ ID NO: 155) was used as the antibody L chain. Antibodiescontaining the light chain of IL6R-L and the above-described heavy chainvariants were expressed and purified according to the method describedin Reference Example 1. The purified antibodies were assessed for theirbinding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV)by the method described in Reference Example 2.

The KD value of each variant to each FcγR is shown in Table 32. In thetable, “alteration” refers to an alteration introduced into IL6R-BP230.IL6R-B3/IL6R-L which is used as the template to produce IL6R-BP230 isindicated by asterisk (*). KD (IIb) of parent polypeptide/KD (IIb) ofaltered polypeptide in the table shows the value obtained by dividingthe KD value of IL6R-B3/IL6R-L for FcγRIIb by the KD value of eachvariant for FcγRIIb. Meanwhile, KD (IIaR) of parent polypeptide/KD(IIaR) of altered polypeptide shows the value obtained by dividing theKD value of IL6R-B3/IL6R-L for FcγR IIaR by the KD value of each variantfor FcγRIIaR. KD (IIaR)/KD (IIb) shows the value obtained by dividingthe KD value of each variant for FcγRIIaR by the KD value of the variantfor FcγRIIb. The greater the value, the higher the selectivity toFcγRIIb. In Table 32, the numeral in the gray-filled cells indicatesthat the binding of FcγR to IgG was concluded to be too weak to analyzecorrectly by kinetic analysis and thus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

TABLE 32

Both FcγRIIb-binding activity and FcγRIIb selectivity ofIL6R-BP264/IL6R-L, IL6R-BP465/IL6R-L, IL6R-BP466/IL6R-L, and IL6R-BP470,resulting from introducing alterations H268E, E333K, E333R, and E333T,respectively, into IL6R-BP230/IL6R-L were increased as compared to thoseof IL6R-BP230/IL6R-L. The FcγRIIb selectivity of IL6R-BP391/IL6R-Lintroduced with the 1332T alteration was reduced while itsFcγRIIb-binding activity was increased as compared to IL6R-BP230/IL6R-L.

Reference Example 21 Exhaustive Introduction of Alterations at AminoAcid Residues Around Position 271 (EU Numbering)

In the structural comparison between Fc (P208)/FcγRIIb and Fc(P238D)/FcγRIIb, the most significant difference is found in thestructure around position 271 (EU numbering) in the CH2 domain B of theFc region (FIG. 48). As described in Reference Example 16, it issuggested that, when, in the structure of Fc (P238D)/FcγRIIb, Asp atposition 270 (EU numbering) forms a tight electrostatic interaction withArg at position 131 in FcγRIIb, the interaction can inducestereochemical stress at Pro at position 271 (EU numbering). In thestructure of Fc (P208)/FcγRIIb, due to the substitution of Gly for Proat position 271 (EU numbering), a positional deviation occurred at themain chain level so as to eliminate the structural distortion, resultingin a large structural change around position 271. There is a possibilitythat additional stabilization of the structure changed around position271 further reduces the entropic energy loss caused by the binding uponformation of an electrostatic interaction with Arg at position 131 inFcγRIIb. Thus, alterations that enhance the FcγRIIb binding or increasethe FcγRIIb selectivity of the Fc region were sought by exhaustiveintroduction of alterations at amino acid residues around position 271(EU numbering).

IL6R-BP267 was constructed as a template in exhaustive introduction ofalterations by introducing alterations E233D, G237D, P238D, H268E, andP271G into IL6R-B3 (SEQ ID NO: 164). IL6R-L (SEQ ID NO: 155) was used asthe antibody L chain. Antibodies containing the light chain of IL6R-Land the above-described heavy chain variants were expressed and purifiedaccording to the method described in Reference Example 1. The purifiedantibodies were assessed for their binding to each FcγR (FcγRIa,FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV) by the method described inReference Example 2. The amino acids at positions 264, 265, 266, 267,269, and 272 (EU numbering) in IL6R-BP267 were substituted with each of18 types of amino acids, except Cys and the amino acid prior tosubstitution. IL6R-L (SEQ ID NO: 155) was used as the antibody L chain.Antibodies containing the light chain of IL6R-L and the above-describedheavy chain variants were expressed and purified according to the methoddescribed in Reference Example 1. The purified antibodies were assessedfor their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, orFcγRIIIaV) by the method described in Reference Example 2. Variantswhose FcγRIIb binding has been enhanced or FcγRIIb selectivity has beenincreased as compared to the FcγRIIb binding or FcγRIIb selectivity ofIL6R-BP267/IL6R-L prior to introduction of the alterations are shown inTable 33.

TABLE 33

The KD value of each variant to each FcγR is shown in Table 33. In thetable, “alteration” refers to an alteration introduced into IL6R-BP267,which was used as a template. IL6R-B3/IL6R-L which is used as thetemplate to produce IL6R-BP267 is indicated by asterisk (*). In thetable, KD (IIb) of parent polypeptide/KD (IIb) of altered polypeptideshows the value obtained by dividing the KD value of IL6R-B3/IL6R-L forFcγRIIb by the KD value of each variant for FcγRIIb. Meanwhile, KD(IIaR) of parent polypeptide/KD (IIaR) of altered polypeptide shows thevalue obtained by dividing the KD value of IL6R-B3/IL6R-L for FcγRIIaRby the KD value of each variant for FcγRIIaR. KD (IIaR)/KD (IIb) showsthe value obtained by dividing the KD value of each variant for FcγRIIaRby the KD value of the variant for FcγRIIb. The greater the value, thehigher the selectivity to FcγRIIb. In Table 33, the numeral in thegray-filled cells indicates that the binding of FcγR to IgG wasconcluded to be too weak to analyze correctly by kinetic analysis andthus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

All the binding activities of variants shown in Table 33 to FcγRIa,FcγRIIaH, and FcγRIIIaV were comparable or reduced as compared to thatof IL6R-B3/IL6R-L. Meanwhile, the FcγRIIb-binding activity of variantsresulting from adding alterations S267A, V264I, E269D, S267E, V266F,S267G, and V266M, respectively, to IL6R-BP267/IL6R-L were increased ascompared to that of IL6R-BP267/IL6R-L prior to addition of alteration.Meanwhile, the KD (IIaR)/KD (IIb) values of variants resulting fromadding the S267A, S267G, E272M, E272Q, D265E, E272D, E272N, V266L,E272I, and E272F alterations, respectively, to IL6R-BP267/IL6R-L wereincreased as compared to that of IL6R-BP267/IL6R-L prior to addition ofalteration. This demonstrates that the S267A, S267G, E272M, E272Q,D265E, E272D, E272N, V266L, E272I, and E272F alterations produce theeffect to improve the FcγRIIb selectivity.

Reference Example 22 Enhancement of the FcγRIIb Binding by Introductionof Alterations into CH3 Region

A substitution alteration of Leu for Pro at position 396 (EU numbering)has been reported to enhance the FcγRIIb binding (Cancer Res. (2007) 67,8882-8890). The amino acid at position 396 (EU numbering) is present ata position which is not directly involved in the interaction with FcγR.However, the amino acid can be assumed to have an effect on theinteraction with FcγR by changing the antibody structure. Thus, thepresent inventors assessed whether the FcγRIIb binding of the Fc regionis enhanced or its FcγRIIb selectivity is increased by exhaustiveintroduction of amino acid alterations at position 396 (EU numbering) inthe Fc region.

IL6R-BP423 was constructed as a template in exhaustive introduction ofalterations by introducing alterations E233D, G237D, P238D, S267A,H268E, P271G, and A330R into IL6R-B3 (SEQ ID NO: 164). Variants, inwhich the amino acid at position 396 (EU numbering) in IL6R-BP423 wassubstituted with each of 18 types of amino acids, except Cys and theamino acid prior to substitution, were constructed. IL6R-L (SEQ ID NO:155) was used as the antibody L chain. Antibodies containing the lightchain of IL6R-L and the above-described heavy chain variants wereexpressed and purified according to the method described in ReferenceExample 1. The purified antibodies were assessed for their binding toeach FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV) by themethod described in Reference Example 2. The binding of the resultingvariants to each FcγR is shown in Table 34.

TABLE 34

In the table, “alteration introduced into IL6R-BP423” refers to analteration introduced into IL6R-BP423, which was used as a template.IL6R-B3/IL6R-L which is used as the template to produce IL6R-BP423 isindicated by asterisk (*). In the table, KD (IIb) of parentpolypeptide/KD (IIb) of altered polypeptide shows the value obtained bydividing the KD value of IL6R-B3/IL6R-L for FcγRIIb by the KD value ofeach variant for FcγRIIb. Meanwhile, KD (IIaR) of parent polypeptide/KD(IIaR) of altered polypeptide shows the value obtained by dividing theKD value of IL6R-B3/IL6R-L for FcγRIIaR by the KD value of each variantfor FcγRIIaR. KD (IIaR)/KD (IIb) shows the value obtained by dividingthe KD value of each variant for FcγRIIaR by the KD value of the variantfor FcγRIIb. The greater the value, the higher the selectivity toFcγRIIb. In Table 34, the numeral in the gray-filled cells indicatesthat the binding of FcγR to IgG was concluded to be too weak to analyzecorrectly by kinetic analysis and thus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

The result shown in Table 34 demonstrates that: the FcγRIIb-bindingactivity of IL6R-BP456/IL6R-L resulting from introducing alterationP396M into IL6R-BP423/IL6R-L, IL6R-BP455/IL6R-L resulting fromintroducing alteration P396L into IL6R-BP423/IL6R-L, IL6R-BP464/IL6R-Lresulting from introducing alteration P396Y into IL6R-BP423/IL6R-L,IL6R-BP450/IL6R-L resulting from introducing alteration P396F intoIL6R-BP423/IL6R-L, IL6R-BP448/IL6R-L resulting from introducingalteration P396D into IL6R-BP423/IL6R-L, IL6R-BP458/IL6R-L resultingfrom introducing alteration P396Q into IL6R-BP423/IL6R-L,IL6R-BP453/IL6R-L resulting from introducing alteration P396I intoIL6R-BP423/IL6R-L, IL6R-BP449/IL6R-L resulting from introducingalteration P396E into IL6R-BP423/IL6R-L, IL6R-BP454/IL6R-L resultingfrom introducing alteration P396K into IL6R-BP423/IL6R-L, andIL6R-BP459/IL6R-L resulting from introducing alteration P396R intoIL6R-BP423/IL6R-L was all increased as compared to that ofIL6R-BP423/IL6R-L prior to introduction of the alterations. Meanwhile,the KD (IIaR)/KD (IIb) value of IL6R-BP456/IL6R-L resulting fromintroducing alteration P396M into IL6R-BP423/IL6R-L was larger ascompared to that of IL6R-BP423/IL6R-L prior to introduction of thealteration, demonstrating the improved FcγRIIb selectivity. As seen inTable 34, the binding activity of the prepared variants to FcγRIa,FcγRIIaH, and FcγRIIIaV was all lower than that of IL6R-B3/IL6R-L, whichwas the parent polypeptide.

Reference Example 23 Preparation of Variants with Enhanced FcγRIIbBinding Using Subclass Sequences

The FcγR binding profile varies depending on the subclass of human IgG.The present inventors assessed whether the difference in the bindingactivity to each FcγR between IgG1 and IgG4 could be utilized toincrease the FcγRIIb-binding activity and/or improve the selectivity.First, IgG1 and IgG4 were analyzed for their binding activity to eachFcγR. IL6R-G4d (SEQ ID NO: 165) containing G4d was constructed as theantibody H chain. G4d is an Fc region that lacks the C-terminal Gly andLys and contains a substitution of Pro for Ser at position 228 (EUnumbering) in human IgG4. IL6R-L (SEQ ID NO: 155) was used as theantibody L chain. Antibodies containing the light chain of IL6R-L andthe heavy chain of IL6R-G1d or IL6R-G4d were expressed and purifiedaccording to the method described in Reference Example 1. The purifiedantibodies were assessed for their binding to each FcγR (FcγRIa,FcγRIIaH, FcγRIIaR, FcγRIIb, or FcγRIIIaV) by the method described inReference Example 2. The binding of the resulting variants to each FcγRis summarized in Table 35.

TABLE 35 KD KD KD KD KD AGAINST AGAINST AGAINST AGAINST AGAINST VARIANTFcγRIa FcγRIIaR FcγRIIaH FcγRIIb FcγRIIIaV NAME (mol/L) (mol/L) (mol/L)(mol/L) (mol/L) IL6R-G1d/ 1.20E−10 9.70E−07 6.50E−07 3.90E−06 4.20E−07IL6R-L IL6R-G4d/ 6.60E−10 2.10E−06 3.40E−06 2.60E−06 3.40E−06 IL6R-L

It was demonstrated that the FcγRIIb binding of IL6R-G4d/IL6R-L was 1.5times stronger than that of IL6R-G1d/IL6R-L whereas the FcγRIIaR bindingof IL6R-G4d/IL6R-L was 2.2 times weaker than that of IL6R-G1d/IL6R-L.Meanwhile, the binding activity of IL6R-G4d/IL6R-L to FcγRIa, FcγRIIaH,and FcγRIIIaV was lower than that of IL6R-G1d/IL6R-L. The resultdescribed above revealed that IL6R-G4d had preferable characteristics ascompared to IL6R-G1d in terms of both FcγRIIb-binding activity andselectivity.

FIG. 53 is an alignment to compare the sequences from CH1 to the Cterminus (positions 118 to 445 (EU numbering)) of G1d and G4d. In FIG.53, amino acid residues that are different between G1d and G4d are boxedwith thick line. The present inventors assessed whether the FcγRIIbbinding could be further increased and/or the FcγRIIb selectivity couldbe further improved by selecting, from the above-described differentamino acids, some portions that are predicted to be involved in theinteraction with FcγR, and grafting at least one amino acid residue ormore of the G4d sequence, which confers a property preferable from theviewpoint of both FcγRIIb-binding activity and selectivity, to a variantwith enhanced FcγRIIb binding.

Specifically, the present inventors produced:

IL6R-BP473 resulting from introducing alteration A327G into IL6R-BP230;IL6R-BP472 resulting from introducing alteration A330S into IL6R-BP230;IL6R-BP471 resulting from introducing alteration P331S into IL6R-BP230;IL6R-BP474 resulting from introducing alterations A330S and P331S intoIL6R-BP230;IL6R-BP475 resulting from introducing alterations A327G and A330S intoIL6R-BP230;IL6R-BP476 resulting from introducing alterations A327G, A330S, andP331S into IL6R-BP230; andIL6R-BP477 resulting from introducing alterations A327G and P331S intoIL6R-BP230.Furthermore, to construct IL6R-BP478, the amino acids from Ala atposition 118 to Thr at position 225 (EU numbering) in IL6R-BP230 wassubstituted with the amino acids from Ala at position 118 to Pro atposition 222 (EU numbering) in G4d. IL6R-L (SEQ ID NO: 155) was used asthe antibody L chain. Antibodies containing the light chain of IL6R-Land the heavy chain variants described above were purified according tothe method described in Reference Example 1. The purified antibodieswere assessed for their binding activity to each FcγR (FcγRIa, FcγRIIaH,FcγRIIaR, FcγRIIb, or FcγRIIIaV) by the method described in ReferenceExample 2.

The KD value of each variant to each FcγR is shown in Table 36. KD (IIb)of parent polypeptide/KD (IIb) of altered polypeptide in the table showsthe value obtained by dividing the KD value of IL6R-B3/IL6R-L forFcγRIIb by the KD value of each variant for FcγRIIb. In the table,“alteration introduced into IL6R-BP230” refers to an alterationintroduced into IL6R-BP230. IL6R-B3/IL6R-L used as the template toproduce IL6R-BP230 is indicated by *1. Meanwhile, IL6R-BP478, in whichthe segment from Ala at position 118 up to Pro at position 222 (EUnumbering) in G4d has been substituted for the segment from Ala atposition 118 up to Thr at position 225 (EU numbering) in IL6R-BP230, isindicated by *2. KD (IIaR) of parent polypeptide/KD (IIaR) of alteredpolypeptide shows the value obtained by dividing the KD value ofIL6R-B3/IL6R-L for FcγR IIaR by the KD value of each variant for FcγRIIaR. KD (IIaR)/KD (IIb) shows the value obtained by dividing the KDvalue of each variant for FcγRIIaR by the KD value of the variant forFcγRIIb. The greater the value, the higher the selectivity to FcγRIIb.In Table 36, the numeral in the gray-filled cells indicates that thebinding of FcγR to IgG was concluded to be too weak to analyze correctlyby kinetic analysis and thus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

TABLE 36

Among the variants shown in Table 36, IL6R-BP473/IL6R-L introduced withalteration A327G had 1.2 times enhanced FcγRIIb binding as compared tothat of IL6R-BP230/IL6R-L. Regarding IL6R-BP478/IL6R-L, resulting fromsubstituting the amino acids of Ala at position 118 up to Thr atposition 225 (EU numbering) in IL6R-BP230 with the amino acids of Ala atposition 118 up to Pro at position 222 (EU numbering) in G4d, itsFcγRIIb binding is enhanced by 1.1 times as compared to that ofIL6R-BP230/IL6R-L, while FcγRIIaR binding of IL6R-BP478/IL6R-L isreduced by 0.9 times as compared to that of IL6R-BP230/IL6R-L. All thevariants also showed lower binding activity to FcγRIa, FcγRIIaH, andFcγRIIIaV as compared to parent polypeptide IL6R-B3/IL6R-L.

Reference Example 24 Assessment of Combinations of Alterations thatEnhance the FcγRIIb Binding or Improve the FcγRIIb Selectivity

Additional combinations of the alterations described herein in thesections up to and including “Reference Example 23”, which alterationshad been found to be effective in the aspect of enhancement of theFcγRIIb binding or the improvement of the FcγRIIb selectivity, wereassessed. Specifically, the alterations that had been assessed to beeffective in enhancing the FcγRIIb binding and/or improving the FcγRIIbselectivity were introduced in combination into IL6R-B3 (SEQ ID NO:164). Furthermore, existing alterations S267E and L328F that enhance theFcγRIIb binding (Seung et al., (Mol. Immunol. (2008) 45, 3926-3933))were introduced into IL6R-B3 to produce IL6R-BP253 as a comparisoncontrol. IL6R-L (SEQ ID NO: 155) was used as the antibody L chain.Antibodies containing the light chain of IL6R-L and the above-describedheavy chain variants were expressed and purified according to the methodas described in Reference Example 1. The purified antibodies wereassessed for their binding to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR,FcγRIIb, or FcγRIIIaV) by the method described in Reference Example 2.

The KD value of each variant to each FcγR is shown in Table 37. In thetable, “alteration” refers to an alteration introduced into IL6R-B3 (SEQID NO: 164). IL6R-B3/IL6R-L which is used as the template to produceeach variant is indicated by asterisk (*). KD (IIb) of parentpolypeptide/KD (IIb) of altered polypeptide shows the value obtained bydividing the KD value of IL6R-B3/IL6R-L for FcγRIIb by the KD value ofeach variant for FcγRIIb. Meanwhile, KD (IIaR) of parent polypeptide/KD(IIaR) of altered polypeptide shows the value obtained by dividing theKD value of IL6R-B3/IL6R-L for FcγR IIaR by the KD value of each variantfor FcγRIIaR. KD (IIaR)/KD (IIb) shows the value obtained by dividingthe KD value of each variant for FcγRIIaR by the KD value of the variantfor FcγRIIb. The greater the value, the higher the selectivity toFcγRIIb as compared to FcγRIIaR. Meanwhile, KD (IIaH)/KD (IIb) shows thevalue obtained by dividing the KD value of each variant for FcγRIIaH bythe KD value of the variant for FcγRIIb. The greater the value, thehigher the selectivity to FcγRIIb as compared to FcγRIIaH. In Table 37,the numeral in the gray-filled cells indicates that the binding of FcγRto IgG was concluded to be too weak to analyze correctly by kineticanalysis and thus was calculated using:

KD=C·R _(max)/(R _(eq)−RI)−C  [Equation 2]

described in Reference Example 2.

TABLE 37

Among the variants shown in Table 37, IL6R-BP253/IL6R-L added with theexisting alterations that enhance the FcγRIIb binding exhibited FcγRIIb-and FcγRIIaR-binding activities increased to 277 times and 529 timesthose of IL6R-B3/IL6R-L prior to introduction of the alterations,respectively. Furthermore, the FcγRIa-binding activity ofIL6R-BP253/IL6R-L was also greater than that of IL6R-B3/IL6R-L.Meanwhile, the FcγRIIaH binding and FcγRIIIaV binding ofIL6R-BP253/IL6R-L were reduced as compared to those of IL6R-B3/IL6R-L.Among other variants, IL6R-BP436/IL6R-L and IL6R-BP438/IL6R-L showed anFcγRIa binding slightly enhanced as compared to that of IL6R-B3/IL6R-Lprior to introduction of the alterations. All other variants showed areduced FcγRIa binding. In addition, all the variants exhibited reducedFcγRIIaH binding and FcγRIIIaV binding as compared to those ofIL6R-B3/IL6R-L.

Regarding IL6R-BP489/IL6R-L, IL6R-BP487/IL6R-L, IL6R-BP499/IL6R-L,IL6R-BP498/IL6R-L, IL6R-BP503/IL6R-L, IL6R-BP488/IL6R-L,IL6R-BP490/IL6R-L, IL6R-BP445/IL6R-L, IL6R-BP507/IL6R-L,IL6R-491/IL6R-L, IL6R-BP506/IL6R-L, IL6R-BP511/IL6R-L,IL6R-BP502/IL6R-L, IL6R-BP510/IL6R-L, IL6R-BP497/IL6R-L,IL6R-BP436/IL6R-L, IL6R-BP423/IL6R-L, IL6R-BP440/IL6R-L,IL6R-BP429/IL6R-L, IL6R-BP438/IL6R-L, IL6R-BP426/IL6R-L,IL6R-BP437/IL6R-L, IL6R-BP439/IL6R-L, IL6R-BP494/IL6R-L,IL6R-BP425/IL6R-L, and IL6R-BP495/IL6R-L, their FcγRIIb binding wasstronger than that of IL6R-BP253/IL6R-L added with the existingalterations that enhance the FcγRIIb binding. Among these, when takingthe binding of IL6R-B3/IL6R-L as 1, the enhancement level ranges from321 times to 3100 times, corresponding to from IL6R-BP495/IL6R-L whichshowed the weakest FcγRIIb binding to IL6R-BP489/IL6R-L which showed thestrongest binding.

The KD (IIaR)/KD (IIb) value of IL6R-BP479/IL6R-L, which was the lowest,was 16.1, while the value of IL6R-BP493/IL6R-L, which was the highest,was 52.1. Thus, the values of the two variants are higher than 0.2 ofIL6R-BP253/IL6R-L. Meanwhile, the KD (IIaH)/KD (IIb) value ofIL6R-BP480/IL6R-L, which was the lowest, was 107.7, while the value ofIL6R-BP426/IL6R-L, which was the highest, was 8362. Thus, the values ofthe two variants are higher than 107.1 of IL6R-BP253/IL6R-L. The resultsdescribed above demonstrate that the FcγRIIb-binding activity of all thevariants shown in Table 37 has been increased as compared to thevariants added with the existing alterations that enhance the FcγRIIbbinding. Furthermore, regardless of whether the FcγR IIa is FcγR IIaR orFcγR IIaH, the FcγRIIb selectivity of the variants shown in Table 37 hasbeen improved relative to the variants added with the existingalterations.

Reference Example 25 Acquisition of Antibodies that Bind to IL-6Receptor in Ca-Dependent Manner from a Human Antibody Library UsingPhage Display Technology (25-1) Preparation of a Phage Display Libraryfor Naive Human Antibodies

A phage display library for human antibodies, consisting of multiplephages presenting the Fab domains of mutually different human antibodysequences, was constructed according to a method known to those skilledin the art using a poly A RNA prepared from human PBMC, and commercialhuman poly A RNA as a template.

(25-2) Acquisition of Antibody Fragments that Bind to Antigen inCa-Dependent Manner from the Library by Bead Panning

The constructed phage display library for naive human antibodies wassubjected to initial selection through concentration of only antibodyfragments having an antigen (IL-6 receptor)-binding ability orconcentration of antibody fragments using a Ca concentration-dependentantigen (IL-6 receptor)-binding ability as an indicator. Concentrationof antibody fragments using a Ca concentration-dependent antigen (IL-6receptor)-binding ability as an indicator were conducted through elutionof the phage library phages bound to IL-6 receptor in the presence of Caions with EDTA that chelates the Ca ions Biotinylated IL-6 receptor wasused as an antigen.

Phages were produced from Escherichia coli carrying the constructedphage display phagemid. A phage library solution was obtained bydiluting with TBS a phage population precipitated by adding 2.5 MNaCl/10% PEG to the E. coli culture solution in which the phages wereproduced. Subsequently, BSA and CaCl₂ were added to the phage librarysolution at a final concentration of 4% BSA and 1.2 mM of calcium ionconcentration. A common panning method using an antigen immobilized onmagnetic beads was referred to as a panning method (J. Immunol. Methods.(2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203;Biotechnol. Prog. (2002) 18(2) 212-20; Mol. Cell. Proteomics (2003) 2(2), 61-9). NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin) were used as magnetic beads.

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution to allow the contact of said phagelibrary solution with the antigen at room temperature for 60 minutes.Magnetic beads, blocked with BSA, were added to be bound toantigen-phage complexes at room temperature for 15 minutes. The beadswere washed once with 1 mL of 1.2 mM CaCl₂/TBS (TBS containing 1.2 mMCaCl₂). Subsequently, a phage solution was recovered by a generalelution method to concentrate an antibody fragment having an IL-6receptor-binding ability, or by elution from beads suspended in 2 mMEDTA/TBS (TBS containing 2 mM EDTA) to concentrate an antibody fragmentusing an IL-6 receptor-binding ability in a Ca concentration-dependentmanner as an indicator. The recovered phage solution was added to 10 mLof the E. coli strain TG1 in a logarithmic growth phase (OD600 of0.4-0.7). The E. coli was cultured with gentle stirring at 37° C. for 1hour to allow the phages to infect the E. coli. The infected E. coli wasinoculated into a 225 mm×225 mm plate. Subsequently, the phages wererecovered from the culture medium of the E. coli after inoculation toprepare a phage library solution.

In the second and subsequent panning, the phages were enriched using theCa-dependent binding ability as an indicator. Specifically, 40 pmol ofthe biotin-labeled antigen was added to the prepared phage librarysolution to allow the contact of the phage library with the antigen atroom temperature for 60 minutes. Magnetic beads, blocked with BSA, wereadded to be bound to antigen-phage complexes at room temperature for 15minutes. The beads were washed with 1 mL of 1.2 mM CaCl₂/TBST and 1.2 mMCaCl₂/TBS. Subsequently, the beads, to which 0.1 mL of 2 mM EDTA/TBS wasadded, were suspended at room temperature. Immediately after that, thebeads were separated using a magnetic stand to collect a phage solution.The recovered phage solution was added to 10 mL of the E. coli strainTG1 in a logarithmic growth phase (OD600 of 0.4-0.7). The E. coli wascultured with gentle stirring at 37° C. for 1 hour to allow the phagesto infect the E. coli. The infected E. coli was inoculated into a 225mm×225 mm plate. Subsequently, the phages were recovered from theculture medium of the E. coli after inoculation to collect a phagelibrary solution. The panning using the Ca-dependent binding ability asan indicator was repeated several times.

(25-3) Examination by Phage ELISA

A phage-containing culture supernatant was collected according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from a singlecolony of E. coli, obtained as described above.

A culture supernatant containing phages, to which BSA and CaCl₂ wereadded at a final concentration of 4% BSA and 1.2 mM of calcium ionconcentration was subjected to ELISA as described below. A StreptaWell96 microtiter plate (Roche) was coated overnight with 100 μL of PBScontaining the biotin-labeled antigen. Each well of said plate waswashed with PBST to remove the antigen, and then the wells were blockedwith 250 μL of 4% BSA-TBS for 1 hour or longer. Said plate with theprepared culture supernatant added to each well, from which the 4%BSA-TBS was removed, was allowed to stand undisturbed at 37° C. for 1hour, allowing the binding of phage-presenting antibody to the antigenpresent in each well. To each well washed with 1.2 mM CaCl₂/TBST, 1.2 mMCaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was allowed to standundisturbed for 30 minutes at 37° C. for incubation. After washing with1.2 mM CaCl₂/TBST, an HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS at a final concentration of 4% BSAand 1.2 mM of ionized calcium concentration was added to each well, andthe plate was incubated for 1 hour. After washing with 1.2 mMCaCl₂/TBST, the chromogenic reaction of the solution in each well with aTMB single solution (ZYMED) added was stopped by adding sulfuric acid.Subsequently, said developed color was measured by measuring absorbanceat 450 nm.

As a result of the above phage ELISA, the base sequence of a geneamplified with specific primers and an antibody fragment identified ashaving a Ca-dependent antigen-binding ability as a template wasanalyzed.

(25-4) Antibody Expression and Purification

As a result of the above phage ELISA, a clone identified as having aCa-dependent antigen-binding ability was introduced into an expressionplasmid for animal cells. Antibodies were expressed as described below.FreeStyle 293-F strain (Invitrogen) derived from human fetal kidneycells was suspended in FreeStyle 293 Expression Medium (Invitrogen),followed by inoculation of 3 mL into each well of a 6-well plate at acell density of 1.33×10⁶ cells/mL. The prepared plasmid was introducedinto the cells by lipofection. The cells were cultured for 4 days in aCO₂ incubator (37° C., 8% CO₂, 90 rpm). Antibodies were purified fromthe culture supernatant obtained above by a method known in the artusing rProtein A Sepharose™ Fast Flow (Amersham Biosciences). Absorbanceof the purified antibody solution was measured at 280 nm using aspectrophotometer. Antibody concentration was calculated from themeasurements obtained using an extinction coefficient calculated by thePACE method (Protein Science (1995) 4, 2411-2423).

Reference Example 26 Examination of Ca-Dependent Binding Ability of theObtained Antibodies to Human IL-6 Receptor

To examine whether or not the binding activities of antibodies6RL#9-IgG1 [heavy chain SEQ ID NO: 117; light chain SEQ ID NO: 118] andFH4-IgG1 [heavy chain SEQ ID NO: 115; light chain SEQ ID NO: 116],obtained in Reference Example 25, to human IL-6 receptor areCa-dependent, the kinetic analysis of the antigen-antibody reactions ofthese antibodies with human IL-6 receptor was conducted using BiacoreT100 (GE Healthcare). H54/L28-IgG1 [heavy chain SEQ ID NO: 113; lightchain SEQ ID NO: 114], described in WO 2009/125825, was used as acontrol antibody that has no Ca-dependent binding activity to human IL-6receptor. The kinetic analysis of the antigen-antibody reactions wasconducted in solutions with 2 mM and 3 μM calcium ion concentrations,set as high and low calcium ion concentration conditions, respectively.The antibody of interest was captured on Sensor chip CM4 (GE Healthcare)on which an appropriate amount of Protein A (Invitrogen) was immobilizedby an amine coupling method. Two buffers [10 mM ACES, 150 mM NaCl, 0.05%(w/v) Tween 20, and 2 mM CaCl₂ (pH 7.4) or 10 mM ACES, 150 mM NaCl,0.05% (w/v) Tween 20, and 3 μmol/L CaCl₂ (pH 7.4)] were used as runningbuffers. These buffers were used for diluting human IL-6 receptor. Allthe measurements were conducted at 37° C.

In the kinetic analysis of antigen-antibody reaction using H54L28-IgG1antibody, the H54L28-IgG1 antibody captured on the sensor chip wasallowed to interact with IL-6 receptor by injecting a diluent of IL-6receptor and running buffer (blank) at a flow rate of 20 μL/min for 3minutes. Subsequently, after the dissociation of IL-6 receptor wasobserved using running buffer at a flow rate of 20 μL/min for 10minutes, the sensor chip was regenerated by injecting 10 mM glycine-HCl(pH 1.5) at a flow rate 30 μL/min for 30 seconds. Kinetics parameters,binding rate constant (ka) (1/Ms) and dissociation rate constant (kd)(1/s), were calculated from the sensorgrams obtained in the measurement.These values were used to calculate the dissociation constant (KD) (M)of the H54L28-IgG1 antibody for human IL-6 receptor. Each parameter wascalculated using the Biacore T100 Evaluation Software (GE Healthcare).

In the kinetic analysis of antigen-antibody reaction using FH4-IgG1 and6RL#9-IgG1 antibodies, the FH4-IgG1 or 6RL#9-IgG1 antibody captured onthe sensor chip was allowed to interact with IL-6 receptor by injectinga diluent of IL-6 receptor and running buffer (blank) at a flow rate of5 μL/min for 15 minutes. Subsequently, the sensor chip was regeneratedby injecting 10 mM glycine-HCl (pH 1.5) at a flow rate 30 μL/min for 30seconds. Dissociation constants (KD) (M) were calculated from thesensorgrams obtained in the measurement, using a steady-state affinitymodel. Each parameter was calculated using the Biacore T100 EvaluationSoftware (GE Healthcare).

The dissociation constants (KD) between each antibody and IL-6 receptorin the presence of 2 mM CaCl₂, determined by the above method, are shownin Table 38.

TABLE 38 ANTIBODY H54/L28-IgG1 FH4-IgG1 6RL#9-IgG1 kD(M) 1.9E−9 5.9E−72.6E−7

The KD value of the H54/L28-IgG1 antibody under the condition of 3 μM Caconcentration can be calculated in the same manner as in the presence of2 mM Ca concentration. Under the condition of 3 μM Ca concentration,FH4-IgG1 and 6RL#9-IgG1 antibodies were barely observed to be bound toIL-6 receptor, thus the calculation of KD values by the method describedabove is difficult. However, the KD values of these antibodies under thecondition of 3 μM Ca concentration can be estimated using Equation 1(Biacore T100 Software Handbook, BR-1006-48, AE 01/2007) describedbelow.

Req=C×Rmax/(KD+C)+RI  [Equation 1]

The meaning of each parameter in the aforementioned [Equation 1] is asfollows:

Req (RU): Steady state binding levelsRmax (RU): Analyte binding capacity of the surfaceRI (RU): Bulk refractive index contribution in the sampleC (M): Analyte concentrationKD (M): Equilibrium dissociation constant

The approximate results of dissociation constant KD values for theantibodies and IL-6 receptor at a Ca concentration of 3 μM, estimatedusing the above-described [Equation 1], are shown in Table 39. In Table39, the Req, Rmax, R1, and C values are estimated based on the assayresult.

TABLE 39 ANTIBODY H54/L28-IgG1 FH4-IgG1 6RL#9-IgG1 Req(RU) 5 10 Rmax(RU)39 72 RI(RU) 0 0 C(M)   5E−06   5E−06 KD(M) 2.2E−9 3.4E−05 3.1E−05

Based on the findings described above, it was predicted that the KDbetween IL-6 receptor and FH4-IgG1 antibody or 6RL#9-IgG1 antibody wasincreased by about 60 or 120 times (the affinity was reduced by 60 or120 times or more) when the concentration of CaCl₂ in the buffer wasdecreased from 2 mM to 3 μM.

Table 40 summarizes the KD values to IL-6 receptor at CaCl₂concentrations of 2 mM and 3 μM and the Ca dependency for the threetypes of antibodies H54/L28-IgG1, FH4-IgG1, and 6RL#9-IgG1.

TABLE 40 ANTIBODY H54/L28-IgG1 FH4-IgG1 6RL#9-IgG1 KD (M) 1.9E−9 5.9E−72.6E−7 (2 mM CaCl₂) KD (M) 2.2E−9 3.4E−5 OR 3.1E−5 OR MORE (3 μM CaCl₂)MORE Ca ABOUT THE ABOUT 60 ABOUT 120 DEPENDENCY SAME TIMES OR TIMES ORMORE MORE

No difference in the binding of the H54/L28-IgG1 antibody to IL-6receptor due to the difference in Ca concentration was observed. On theother hand, the binding of FH4-IgG1 and 6RL#9-IgG1 antibodies to IL-6receptor was observed to be significantly attenuated under the conditionof the low Ca concentration (Table 40).

Reference Example 27 Examination of Calcium Ion Binding to the AntibodyObtained

Subsequently, the intermediate temperature of thermal denaturation (Tmvalue) was measured by differential scanning calorimetry (DSC) as anindicator for examining calcium ion binding to the antibody (MicroCalVP-Capillary DSC, MicroCal). The intermediate temperature of thermaldenaturation (Tm value) is an indicator of stability. The intermediatetemperature of thermal denaturation (Tm value) becomes higher when aprotein is stabilized through calcium ion binding, as compared with nocalcium ion binding (J. Biol. Chem. (2008) 283, 37, 25140-25149). Thebinding activity of calcium ion to antibody was examined by examiningchanges in the Tm value of the antibody depending on the changes in thecalcium ion concentration of the antibody solution. The purifiedantibody was subjected to dialysis (EasySEP, TOMY) using an externalsolution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl₂ (pH 7.4), or 20mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl₂ (pH 7.4). DSC measurement wasconducted at a heating rate of 240° C./hr from 20 to 115° C. using anantibody solution prepared at about 0.1 mg/mL with the dialysate as atest substance. The intermediate temperatures of thermal denaturation(Tm values) of the Fab domains of each antibody, calculated based on thedenaturation curve obtained by DSC, are shown in Table 41.

TABLE 41 CALCIUM ION CONCENTRATION ΔTm (° C.) ANTIBODY 3 μM 2 mM 2 mM −3 μM H54/L28-IgG1 92.87 92.87 0.00 FH4-IgG1 74.71 78.97 4.26 6RL#9-IgG177.77 78.98 1.21

From the results shown in Table 41, it is indicated that the Tm valuesof the Fab of the FH4-IgG1 and 6RL#9-IgG1 antibodies, which show acalcium-dependent binding ability, varied with changes in the calciumion concentration, while the Tm value of the Fab of the H54/L28-IgG1antibody which shows no calcium-dependent binding ability did not varywith changes in the calcium ion concentration. The variation in the Tmvalues of the Fab of the FH4-IgG1 and 6RL#9-IgG1 antibodies demonstratesthat calcium ions bound to these antibodies to stabilize the Fabportions. The above results show that calcium ions bound to the FH4-IgG1and 6RL#9-IgG1 antibodies, while no calcium ion bound to theH54/L28-IgG1 antibody.

Reference Example 28 Identification of Calcium Ion-Binding Site inAntibody 6RL#9 by X-Ray Crystal Structure Analysis (28-1) X-Ray CrystalStructure Analysis

As described in Reference Example 27, the measurements of thermaldenaturation temperature Tm suggested that antibody 6RL#9 binds tocalcium ion. However, it was unpredictable which portion of antibody6RL#9 binds to calcium ion. Then, by using the technique of X-raycrystal structure analysis, residues of antibody 6RL#9 that interactwith calcium ion were identified.

(28-2) Expression and Purification of Antibody 6RL#9

Antibody 6RL#9 was expressed and purified for X-ray crystal structureanalysis. Specifically, animal expression plasmids constructed to becapable of expressing the heavy chain (SEQ ID NO: 117) and light chain(SEQ ID NO: 118) of antibody 6RL#9 were introduced transiently intoanimal cells. The constructed plasmids were introduced by thelipofection method into cells of human fetal kidney cell-derivedFreeStyle 293-F (Invitrogen) suspended in 800 ml of the FreeStyle 293Expression Medium (Invitrogen) (final cell density: 1×10⁶ cells/mL). Theplasmid-introduced cells were cultured in a CO₂ incubator (37° C., 8%CO₂, 90 rpm) for five days. From the culture supernatant obtained asdescribed above, antibodies were purified by a method known to thoseskilled in the art using the rProtein A Sepharose™ Fast Flow (AmershamBiosciences). Absorbance at 280 nm of purified antibody solutions wasmeasured using a spectrophotometer. Antibody concentrations werecalculated from the measured values using an extinction coefficientcalculated by the PACE method (Protein Science (1995) 4, 2411-2423).

(28-3) Purification of Antibody 6RL#9 Fab Fragment

Antibody 6RL#9 was concentrated to 21 mg/ml using an ultrafilter with amolecular weight cutoff of 10,000 MWCO. A 5 mg/mL antibody sample (2.5mL) was prepared by diluting the antibody solution using 4 mML-cysteine/5 mM EDTA/20 mM sodium phosphate buffer (pH 6.5). 0.125 mg ofpapain (Roche Applied Science) was added to the sample. After stirring,the sample was incubated at 35° C. for two hours. After incubation, atablet of Protease Inhibitor Cocktail Mini, EDTA-free (Roche AppliedScience) was dissolved in 10 ml of 25 mM MES buffer (pH 6) and added tothe sample. The sample was incubated on ice to stop the papainproteolytic reaction. Then, the sample was loaded onto a 1-mlcation-exchange column HiTrap SP HP (GE Healthcare) equilibrated with 25mM MES buffer (pH 6), downstream of which a 1-ml HiTrap MabSelect SureProtein A column (GE Healthcare) was connected in tandem. A purifiedfraction of the Fab fragment of antibody 6RL#9 was obtained byperforming elution with a linear NaCl concentration gradient up to 300mM in the above-described buffer. Then, the resulting purified fractionwas concentrated to about 0.8 ml using a 5000 MWCO ultrafilter. Theconcentrate was loaded onto a gel filtration column Superdex 200 10/300GL (GE Healthcare) equilibrated with 100 mM HEPES buffer (pH 8)containing 50 mM NaCl. The purified Fab fragment of antibody 6RL#9 forcrystallization was eluted from the column using the same buffer. Allthe column treatments described above were carried out at a lowtemperature of 6 to 7.5° C.

(28-4) Crystallization of the Antibody 6RL#9 Fab Fragment in thePresence of Ca

Seed crystals of the 6RL#9 Fab fragment were prepared in advance undergeneral conditions. Then, the purified Fab fragment of antibody 6RL#9 in5 mM CaCl₂ was concentrated to 12 mg/ml with a 5000 MWCO ultrafilter.Next, the sample concentrated as described above was crystallized by thehanging drop vapor diffusion method using 100 mM HEPES buffer (pH 7.5)containing 20% to 29% PEG4000 as a reservoir solution. Theabove-described seed crystals were crushed in 100 mM HEPES buffer (pH7.5) containing 29% PEG4000 and 5 mM CaCl₂, and serially diluted to 100to 10,000 folds. Then, 0.2 μL of diluted solutions were combined with amixture of 0.8 μL of the reservoir solution and 0.8 μL of theconcentrated sample to prepare crystallization drops on a glass coverslide. The crystallization drops were allowed to stand at 20° C. for twoto three days to prepare thin plate-like crystals. X-ray diffractiondata were collected using the crystals.

(28-5) Crystallization of the Antibody 6RL#9 Fab Fragment in the Absenceof Ca

The purified Fab fragment of antibody 6RL#9 was concentrated to 15 mg/mlusing a 5000 MWCO ultrafilter. Then, the sample concentrated asdescribed above was crystallized by the hanging drop vapor diffusionmethod using 100 mM HEPES buffer (pH 7.5) containing 18% to 25% PEG4000as a reservoir solution. Crystals of the antibody 6RL#9 Fab fragmentobtained in the presence of Ca were crushed in 100 mM HEPES buffer (pH7.5) containing 25% PEG4000, and serially diluted to 100 to 10,000folds. Then, 0.2 μL of diluted solutions were combined with a mixture of0.8 μL of the reservoir solution and 0.8 μL of the concentrated sampleto prepare crystallization drops on a glass cover slide. Thecrystallization drops were allowed to stand at 20° C. for two to threedays to prepare thin plate-like crystals. X-ray diffraction data werecollected using the crystals.

(28-6) X-Ray Diffraction Data Measurement of Fab Fragment Crystal fromAntibody 6RL#9 in the Presence of Ca

Crystals of the Fab fragment of antibody 6RL#9 prepared in the presenceof Ca were soaked into 100 mM HEPES buffer (pH 7.5) solution containing35% PEG4000 and 5 mM CaCl₂. The single crystal was fished out of theexterior solution using a pin with attached tiny nylon loop, and frozenin liquid nitrogen. X-ray diffraction data of the frozen crystal wascollected from beam line BL-17A of the Photon Factory in the High EnergyAccelerator Research Organization. The frozen crystal was constantlyplaced in a nitrogen stream at −178° C. to maintain in a frozen stateduring the measurement. A total of 180 diffraction images were collectedusing the CCD detector Quantum315r (ADSC) attached to the beam line withrotating the crystal 1° at a time. Lattice constant determination,diffraction spot indexing, and diffraction data analysis were performedusing programs Xia2 (CCP4 Software Suite), XDS Package (WalfgangKabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensitydata up to 2.2 Å resolution was obtained. The crystal belongs to thespace group P2₁2₁2₁ with lattice constant a=45.47 Å, b=79.86 Å, c=116.25Å, α=90°, β=90°, and γ=90°.

(28-7) X-Ray Diffraction Data Measurement of the Fab Fragment Crystalfrom Antibody 6RL#9 in the Absence of Ca

Crystals of the Fab fragment of antibody 6RL#9 prepared in the absenceof Ca were soaked in 100 mM HEPES buffer (pH 7.5) solution containing35% PEG4000. The single crystal was fished out of the exterior solutionusing a pin with attached tiny nylon loop, and frozen in liquidnitrogen. X-ray diffraction data of the frozen crystal was collectedfrom beam line BL-5A of the Photon Factory in the High EnergyAccelerator Research Organization. The frozen crystal was constantlyplaced in a nitrogen stream at −178° C. to maintain in a frozen stateduring the measurement. A total of 180 diffraction images were collectedusing the CCD detector Quantum210r (ADSC) attached to the beam line withrotating the crystal 1° at a time. Lattice constant determination,diffraction spot indexing, and diffraction data analysis were performedusing programs Xia2 (CCP4 Software Suite), XDS Package (WalfgangKabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensitydata up to 2.3 Å resolution was obtained. The crystal belongs to thespace group P2₁2₁2₁ with lattice constant a=45.40 Å, b=79.63 Å, c=116.07Å, α=90°, β=90°, γ=90°, and thus is structurally identical to thecrystal prepared in the presence of Ca.

(28-8) Structural Analysis of the Fab Fragment Crystal from Antibody6RL#9 in the Presence of Ca

The crystal structure of the antibody 6RL#9 Fab fragment in the presenceof Ca was determined by a molecular replacement method using the Phaserprogram (CCP4 Software Suite). The number of molecules in anasymmetrical unit was estimated to be one from the size of crystallattice and molecular weight of the antibody 6RL#9 Fab fragment. Basedon the primary sequence homology, a portion of amino acid positions 112to 220 from A chain and a portion of amino acid positions 116 to 218from B chain in the conformational coordinate of PDB code 1ZA6 were usedas model molecules for analyzing the CL and CH1 regions. Then, a portionof amino acid positions 1 to 115 from B chain in the conformationalcoordinate of PDB code 1 ZA6 was used as a model molecule for analyzingthe VH region. Finally, a portion of amino acid positions 3 to 147 ofthe light chain in the conformational coordinate of PDB code 2A9M wasused as a model molecule for analyzing the VL region. Based on thisorder, an initial structure model for the antibody 6RL#9 Fab fragmentwas obtained by determining from translation and rotation functions thepositions and orientations of the model molecules for analysis in thecrystal lattice. The crystallographic reliability factor R for thereflection data at 25 to 3.0 Å resolution was 46.9% and Free R was 48.6%after rigid body refinement where the VH, VL, CH1, and CL domains wereeach allowed to deviate from the initial structure model. Then, modelrefinement was achieved by repeating structural refinement using programRefmac5 (CCP4 Software Suite) followed by model revision performed usingprogram Coot (Paul Emsley) with reference to the Fo-Fc and 2Fo-Fcelectron density maps where the coefficients Fo-Fc and 2Fo-Fc werecalculated using experimentally determined structural factor Fo,structural factor Fc calculated based on the model, and the phases. Thefinal refinement was carried out using program Refmac5 (CCP4 SoftwareSuite) based on the Fo-Fc and 2Fo-Fc electron density maps by addingwater molecule and Ca ion into the model. With 21,020 reflection data at25 to 2.2 Å resolution, eventually the crystallographic reliabilityfactor R became 20.0% and free R became 27.9% for the model consistingof 3440 atoms.

(28-9) Structural Analysis of the Fab Fragment Crystal from Antibody6RL#9 in the Absence of Ca

The crystal structure of the antibody 6RL#9 Fab fragment in the absenceof Ca was determined based on the structure of the crystal prepared inthe presence of Ca. Water and Ca ion molecules were omitted from theconformational coordinate of the crystal of the antibody 6RL#9 Fabfragment prepared in the presence of Ca. The crystallographicreliability factor R for the data of reflection at 25 to 3.0 Åresolution was 30.3% and Free R was 31.7% after the rigid bodyrefinement where the VH, VL, CH1, and CL domains were each allowed todeviate. Then, model refinement was achieved by repeating structuralrefinement using program Refmac5 (CCP4 Software Suite) followed by modelrevision performed using program Coot (Paul Emsley) with reference tothe Fo-Fc and 2Fo-Fc electron density maps where the coefficients Fo-Fcand 2Fo-Fc were calculated using experimentally determined structuralfactor Fo, structural factor Fc calculated based on the model, and thephases. The final refinement was carried out using program Refmac5 (CCP4Software Suite) based on the Fo-Fc and 2Fo-Fc electron density maps byadding water molecule into the model. With 18,357 reflection data at 25to 2.3 Å resolution, eventually the crystallographic reliability factorR became 20.9% and free R became 27.7% for the model consisting of 3351atoms.

(28-10) Comparison of X-Ray Crystallographic Diffraction Data of the FabFragments of Antibody 6RL#9 Between in the Presence and Absence of Ca

When the crystal structures of the Fab fragments of antibody 6RL#9 arecompared between in the presence and absence of Ca, significant changesare seen in the heavy chain CDR3. The structure of the heavy chain CDR3of the antibody 6RL#9 Fab fragment determined by X-ray crystal structureanalysis is shown in FIG. 54. Specifically, a calcium ion resided at thecenter of the heavy chain CDR3 loop region of the antibody 6RL#9 Fabfragment crystal prepared in the presence of Ca. The calcium ion wasassumed to interact with positions 95, 96, and 100a (Kabat numbering) ofthe heavy chain CDR3. It was believed that the heavy chain CDR3 loopwhich is important for the antigen binding was stabilized by calciumbinding in the presence of Ca, and became an optimum structure forantigen binding. There is no report demonstrating that calcium binds tothe antibody heavy chain CDR3. Thus, the calcium-bound structure of theantibody heavy chain CDR3 is a novel structure.

The calcium-binding motif present in the heavy chain CDR3, revealed inthe structure of the Fab fragment of the 6RL#9 antibody may also becomea new design element for the Ca library for obtaining antigen-bindingdomain included in the antigen-binding molecule of the present inventionwhose antigen-binding activity varies depending on the calcium ionconcentration. The calcium-binding motif was introduced into a lightchain variable region in later-described Reference Examples 38 and 39,and for example, a library containing the heavy chain CDR3 of the 6RL#9antibody and flexible residues in other CDRs including the light chainis thought to be possible.

Reference Example 29 Preparation of Antibodies that Bind to IL-6 in aCa-Dependent Manner from a Human Antibody Library Using Phage DisplayTechniques (29-1) Construction of a Phage Display Library of Naive HumanAntibodies

A human antibody phage display library containing multiple phages thatdisplay various human antibody Fab domain sequences was constructed by amethod known to those skilled in the art using, as a template, polyA RNAprepared from human PBMC, commercially available human polyA RNA, andsuch.

(29-2) Preparation of Antibody Fragments that Bind to the Antigen in aCa-Dependent Manner from Library by Bead Panning

Primary selection from the constructed phage display library of naivehuman antibodies was carried out by enriching antibody fragments thathave antigen (IL-6)-binding activity. The antigen used wasbiotin-labeled IL-6.

Phages were produced from E. coli carrying the constructed phagemid forphage display. To precipitate the phages produced by E. coli, 2.5 MNaCl/10% PEG was added to the E. coli culture medium. The phage fractionwas diluted with TBS to prepare a phage library solution. Then, BSA andCaCl₂ were added the phage library solution at final concentrations of4% BSA and 1.2 mM calcium ion concentration, respectively. The panningmethod used was a conventional panning method using antigen-immobilizedmagnetic beads (J. Immunol. Methods. (2008) 332(1-2): 2-9; J. Immunol.Methods. (2001) 247(1-2): 191-203; Biotechnol. Prog. (2002) 18(2):212-20; Mol. Cell. Proteomics (2003) 2(2): 61-9). The magnetic beadsused were NeutrAvidin-coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin-coated beads (Dynabeads M-280Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution. Thus, the solution was contacted withthe antigen at room temperature for 60 minutes. Magnetic beads blockedwith BSA were added, and the antigen-phage complex was allowed to bindto the magnetic beads at room temperature for 15 minutes. The beads werewashed three times with 1.2 mM CaCl₂/TBST (TBST containing 1.2 mMCaCl₂), and then twice with 1 ml of 1.2 mM CaCl₂/TBS (TBS containing 1.2mM CaCl₂). Thereafter, 0.5 ml of 1 mg/ml trypsin was added to the beads.After 15 minutes of dispersion at room temperature, the beads wereimmediately separated using a magnetic stand to collect a phagesolution. The prepared phage solution was added to 10 ml of E. coli ofstain TG1 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was cultured with gentle stirring at 37° C. for one hour to infectthe phages. The infected E. coli was seeded in a plate (225 mm×225 mm).Then, phages were collected from the culture medium of the seeded E.coli to prepare a phage library solution.

In the second round and subsequent panning, phages were enriched usingthe Ca-dependent binding activity as an indicator. Specifically, 40 pmolof the biotin-labeled antigen was added to the prepared phage librarysolution. Thus, the phage library was contacted with the antigen at roomtemperature for 60 minutes. Magnetic beads blocked with BSA were added,and the antigen-phage complex was allowed to bind to the magnetic beadsat room temperature for 15 minutes. The beads were washed with 1 ml of1.2 mM CaCl₂/TBST and 1.2 mM CaCl₂/TBS. Next, 0.1 ml of 2 mM EDTA/TBSwas added to the beads. After dispersion at room temperature, the beadswere immediately separated using a magnetic stand to collect a phagesolution. The pIII protein (helper phage-derived protein pIII) wascleaved from phages that did not display Fab by adding 5 μl of 100 mg/mltrypsin to the collected phage solution to eliminate the ability ofphages displaying no Fab to infect E. coli. Phages collected from thetrypsinized phage solution were added to 10 ml of E. coli cells of theTG1 strain at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was cultured while gently stirring at 37° C. for one hour to infectphage. The infected E. coli was seeded in a plate (225 mm×225 mm). Then,phages were collected from the culture medium of the seeded E. coli toprepare a phage library solution. Panning was performed three timesusing the Ca-dependent binding activity as an indicator.

(29-3) Assessment by Phage ELISA

Culture supernatants containing phages were collected from singlecolonies of E. coli obtained by the method described above according toa conventional method (Methods Mol. Biol. (2002) 178, 133-145).

BSA and CaCl₂ were added at final concentrations of 4% BSA and 1.2 mMcalcium ion concentration, respectively, to the phage-containing culturesupernatants. The supernatants were subjected to ELISA by the followingprocedure. A StreptaWell 96-well microtiter plate (Roche) was coatedovernight with 100 μl of PBS containing the biotin-labeled antigen. Theantigen was removed by washing each well of the plate with PBST. Then,the wells were blocked with 250 μl of 4% BSA-TBS for one hour or more.After removal of 4% BSA-TBS, the prepared culture supernatants wereadded to the each well. The plate was incubated at 37° C. for one hourso that the antibody-displaying phages were allowed to bind to theantigen on each well. After each well was washed with 1.2 mM CaCl₂/TBST,1.2 mM CaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was left forincubation at 37° C. for 30 minutes. After washing with 1.2 mMCaCl₂/TBST, an HRP-conjugated anti-M13 antibody (Amersham PharmaciaBiotech) diluted with TBS containing BSA and calcium ion at finalconcentrations of 4% BSA and 1.2 mM calcium ion concentration was addedto each well, and the plate was incubated for one hour. After washingwith 1.2 mM CaCl₂/TBST, the TMB single solution (ZYMED) was added toeach well. The chromogenic reaction in the solution of each well wasstopped by adding sulfuric acid. Then, the developed color was assessedby measuring absorbance at 450 nm.

From the isolated 96 clones, antibody 6KC4-1#85 having Ca-dependentIL-6-binding activity was obtained by phage ELISA. Using antibodyfragments that were predicted to have a Ca-dependent antigen-bindingactivity based on the result of the phage ELISA described above as atemplate, genes were amplified with specific primers and their sequenceswere analyzed. The heavy-chain and light-chain variable region sequencesof antibody 6KC4-1#85 are shown in SEQ ID NOs: 119 and 120,respectively. The polynucleotide encoding the heavy-chain variableregion of antibody 6KC4-1#85 (SEQ ID NO: 119) was linked to apolynucleotide encoding an IgG1-derived sequence by PCR method. Theresulting DNA fragment was inserted into an animal cell expressionvector to construct an expression vector for the heavy chain of SEQ IDNO: 121. A polynucleotide encoding the light-chain variable region ofantibody 6KC4-1#85 (SEQ ID NO: 120) was linked to a polynucleotideencoding the constant region of the natural Kappa chain (SEQ ID NO: 54)by PCR. The linked DNA fragment was inserted into an animal cellexpression vector. Sequences of the constructed variants were confirmedby a method known to those skilled in the art.

(29-4) Expression and Purification of Antibodies

Clone 6KC4-1#85 that was predicted to have a Ca-dependentantigen-binding activity based on the result of phage ELISA was insertedinto animal cell expression plasmids. Antibody expression was carriedout by the following method. Cells of human fetal kidney cell-derivedFreeStyle 293-F (Invitrogen) were suspended in the FreeStyle 293Expression Medium (Invitrogen), and plated at a cell density of 1.33×10⁶cells/ml (3 ml) into each well of a 6-well plate. The prepared plasmidswere introduced into cells by a lipofection method. The cells arecultured for four days in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). Fromthe culture supernatants, antibodies were purified using the rProtein ASepharose™ Fast Flow (Amersham Biosciences) by a method known to thoseskilled in the art. Absorbance at 280 nm of the purified antibodysolutions was measured using a spectrophotometer. Antibodyconcentrations were calculated from the determined values using anextinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

Reference Example 30 Assessment of Antibody 6KC4-1#85 for Calcium IonBinding

Calcium-dependent antigen-binding antibody 6KC4-1#85 which was isolatedfrom a human antibody library was assessed for its calcium binding.Whether the measured Tm value varies depending on the ionized calciumconcentration condition was assessed according to the method describedin Reference Example 27.

Tm values for the Fab domain of antibody 6KC4-1#85 are shown in Table42. As shown in Table 42, the Tm value of the 6KC4-1#85 antibody Fabdomain varied depending on the calcium ion concentration. Thisdemonstrates that antibody 6KC4-1#85 binds to calcium.

TABLE 42 CALCIUM ION CONCENTRATION ΔTm (° C.) ANTIBODY 3 μM 2 mM 2 mM −3 μM 6KC4-1#85 71.49 75.39 3.9

Reference Example 31 Identification of Calcium Ion-Binding Site inAntibody 6KC4-1#85

As demonstrated in Reference Example 30, antibody 6KC4-1#85 binds tocalcium ion. However, 6KC4-1#85 does not have a calcium-binding motifsuch as the hVk5-2 sequence which was revealed from assessment to have acalcium-binding motif. Then, whether calcium ion binds to either or bothof the heavy chain and the light chain of antibody 6KC4-1#85 wasconfirmed by assessing the calcium ion binding of altered antibodiesresulting from exchanging the heavy chain and light chain of 6KC4-1#85respectively with those of an anti-glypican 3 antibody (heavy chainsequence GC_H (SEQ ID NO: 55), light chain sequence GC_L (SEQ ID NO:56)) which does not bind calcium ion. The Tm values of alteredantibodies measured according to the method described in ReferenceExample 27 are shown in Table 43. The result suggests that the heavychain of antibody 6KC4-1#85 binds to calcium, because the Tm values ofthe altered antibody having the heavy chain of antibody 6KC4-1#85changed depending on calcium ion concentration.

TABLE 43 CALCIUM ION HEAVY LIGHT CONCENTRATION ΔTm (° C.) CHAIN CHAIN 3μM 2 mM 2 mM − 3 μM 6KC4-1#85 6KC4-1#85 71.46 75.18 3.72 6KC4-1#85 GC_L78.87 80.01 1.14 GC_H 6KC4-1#85 75.69 75.94 0.25 GC_H GC_L 79.94 80.010.07

Thus, to further identify residues responsible for the calcium ionbinding of the heavy chain of antibody 6KC4-1#85, altered heavy chains(6_H1-11 (SEQ ID NO: 126), 6_H1-12 (SEQ ID NO: 127), 6_H1-13 (SEQ ID NO:128), 6_H1-14 (SEQ ID NO: 129), 6_H1-15 (SEQ ID NO: 130)) or alteredlight chains (6_L1-5 (SEQ ID NO: 131) and 6_L1-6 (SEQ ID NO: 132)) wereconstructed by substituting an Asp (D) residue in the CDR of antibody6KC4-1#85 with an Ala (A) residue which does not participate in thebinding or chelation of calcium ion. By the method described inReference Example 29, altered antibodies were purified from the culturemedia of animal cells introduced with expression vectors carrying thealtered antibody genes. The purified altered antibodies were assessedfor their calcium binding according to the method described in ReferenceExample 27. The measurement result is shown in Table 44. As shown inTable 44, substitution of an Ala residue for the residue at position 95or 101 (Kabat numbering) in the heavy chain CDR3 of antibody 6KC4-1#85resulted in loss of the calcium-binding activity of antibody 6KC4-1#85.This suggests that these residues are responsible for calcium binding.The calcium-binding motif located at the base of the CDR3 loop in theheavy chain of antibody 6KC4-1#85, which was found based on the calciumbinding capacity of the antibody altered from antibody 6KC4-1#85, can bea new factor for designing Ca libraries which are used to obtainantigen-binding domains whose antigen-binding activity changes dependingon calcium ion concentration and which are to be contained inantigen-binding molecules of the present invention. In ReferenceExamples 38 and 39 below, calcium-binding motifs were introduced intothe light chain variable region. Meanwhile, such libraries include, forexample, those containing the heavy chain CDR3 from antibody 6KC4-1#85and flexible residues in the CDRs other than the heavy chain CDR3 butincluding the light chain CDRs.

TABLE 44 CALCIUM ION CONCENTRATION ΔTm (° C.) HEAVY CHAIN LIGHT CHAINALTERED RESIDUE 3 μM 2 mM 2 mM − 3 μM 6KC4-1#85 6KC4-1#85 WILD-TYPE71.49 75.39 3.9 6H1-11 6KC4-1#85 H CHAIN 71.73 75.56 3.83 POSITION 61(Kabat NUMBERING) 6H1-12 6KC4-1#85 H CHAIN 72.9 73.43 0.53 POSITION 95(Kabat NUMBERING) 6H1-13 6KC4-1#85 H CHAIN 70.94 76.25 5.31 POSITION100a (Kabat NUMBERING) 6H1-14 6KC4-1#85 H CHAIN 73.95 75.14 1.19POSITION 100g (Kabat NUMBERING) 6H1-15 6KC4-1#85 H CHAIN 65.37 66.250.87 POSITION 101 (Kabat NUMBERING) 6KC4-1#85 6L1-5 L CHAIN 71.92 76.084.16 POSITION 50 (Kabat NUMBERING) 6KC4-1#85 6L1-6 L CHAIN 72.13 78.746.61 POSITION 92 (Kabat NUMBERING)

Reference Example 32 Examination of Effects of Ca-Dependent BindingAntibody on Plasma Retention of Antigen Using Normal Mice (32-1) In VivoTest Using Normal Mice

To a normal mouse (C57BL/6J mouse, Charles River Japan), hsIL-6R(soluble human IL-6 receptor prepared in Reference Example 3) alone wasadministered, or hsIL-6R and anti-human IL-6 receptor antibody wereadministered simultaneously to examine the kinetics of the hsIL-6R andanti-human IL-6 receptor antibody in vivo. A single dose (10 mL/kg) ofthe hsIL-6R solution (5 μg/mL) or a mixture of hsIL-6R and anti-humanIL-6 receptor antibody was administered into the tail vein. The aboveH54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 were used as anti-human IL-6receptor antibodies.

The hsIL-6R concentration in all the mixtures is 5 μg/mL. Theconcentrations of anti-human IL-6 receptor antibody vary with theantibodies: 0.1 mg/mL for H54/L28-IgG1 and 10 mg/mL for 6RL#9-IgG1 andFH4-IgG1. At this time, it was thought that most of the hsIL-6Rs bind tothe antibody because the anti-human IL-6 receptor antibody exists in asufficient or excessive amount for hsIL-6R. Blood samples were collectedat 15 minutes, 7 hours and 1, 2, 4, 7, 14, 21, and 28 days after theadministration. The blood samples obtained were immediately centrifugedfor 15 minutes at 4° C. and 12,000 rpm to separate plasma. The separatedplasma was stored in a freezer set to −20° C. or lower until the time ofmeasurement.

(32-2) Determination of Plasma Anti-Human IL-6 Receptor AntibodyConcentration in Normal Mice by ELISA

The plasma concentration of anti-human IL-6 receptor antibody in a mousewas determined by ELISA. First, Anti-Human IgG (γ-chain specific)F(ab′)2 Fragment of Antibody (SIGMA) was dispensed into a Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International), and was allowed to standundisturbed overnight at 4° C. to prepare an anti-human IgG-immobilizedplate. Calibration curve samples at a plasma concentration of 0.64,0.32, 0.16, 0.08, 0.04, 0.02, or 0.01 μg/mL, and mouse plasmameasurement samples diluted by 100-fold or above were each dispensedinto the anti-human IgG-immobilized plate, followed by incubation for 1hour at 25° C. Subsequently, the plate was allowed to react with abiotinylated anti-human IL-6 R antibody (R&D) for 1 hour at 25° C.,followed by reaction with Streptavidin-PolyHRP80 (StereospecificDetection Technologies) for 0.5 hours at 25° C. The chromogenic reactionwas conducted using TMB One Component HRP Microwell Substrate (BioFXLaboratories) as a substrate. After the chromogenic reaction was stoppedby adding 1N-sulfuric acid (Showa Chemical), absorbance at 450 nm of thecolored solution was measured using a microplate reader. The plasmaconcentration in the mouse was calculated from the absorbance of thecalibration curve using the SOFTmax PRO analysis software (MolecularDevices). Changes in the plasma concentrations of antibodies,H54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1, in the normal mice afterintravenous administration, measured as described above, are shown inFIG. 55.

(32-3) Determination of Plasma hsIL-6R Concentration by anElectrochemiluminescence Method

The plasma concentration of hsIL-6R in a mouse was determined by anelectrochemiluminescence method. A hsIL-6R calibration curve sampleprepared at 2,000, 1,000, 500, 250, 125, 62.5, or 31.25 pg/mL, and amouse plasma measurement sample diluted by 50-fold or above, were mixedwith a monoclonal anti-human IL-6R antibody (R&D) ruthenated withSULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-humanIL-6 R antibody (R&D), and tocilizumab (heavy chain SEQ ID NO: 111,light chain SEQ ID NO: 112), followed by overnight reaction at 4° C. Atthat time, the assay buffer contained 10 mM EDTA to reduce the free Caconcentration in the sample and dissociate almost all the hsIL-6Rs inthe sample from 6RL#9-IgG1 or FH4-IgG1 to be bound to the addedtocilizumab. Subsequently, said reaction solution was dispensed into anMA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, afterwashing each well of the plate that was allowed to react for 1 hour at25° C., Read Buffer T (×4) (Meso Scale Discovery) was dispensed intoeach well. Immediately, the reaction solution was subjected tomeasurement using a SECTOR PR 400 reader (Meso Scale Discovery). Theconcentration of hsIL-6R was calculated from the response of thecalibration curve using the SOFTmax PRO analysis software (MolecularDevices). Changes in the plasma concentration of hsIL-6R in the normalmouse after intravenous administration, determined as described above,are shown in FIG. 56.

As a result, the disappearance of hsIL-6R was very rapid when hsIL-6Rwas administered alone, while the disappearance of hsIL-6R wassignificantly delayed when hsIL-6R was administered simultaneously withH54/L28-IgG1, a conventional antibody having no Ca-dependent bindingability to soluble human IL-6 receptor. In contrast, the disappearanceof hsIL-6R was significantly accelerated when hsIL-6R was administeredsimultaneously with 6RL#9-IgG1 or FH4-IgG1 having 100-fold or higherCa-dependent binding ability to hsIL-6R. The plasma concentrations ofhsIL-6R one day after soluble human IL-6 receptor was administeredsimultaneously with 6RL#9-IgG1 and FH4-IgG1 were reduced 39-fold and2-fold, respectively, as compared with simultaneous administration withH54/L28-IgG1. Thus, the calcium-dependent binding antibodies wereconfirmed to be able to accelerate antigen disappearance from theplasma.

Reference Example 33 Exploration of Human Germline Sequences that Bindto Calcium Ion

(33-1) Antibody that Binds to Antigen in a Calcium-Dependent Manner

Antibodies that bind to an antigen in a calcium-dependent manner(calcium-dependent antigen-binding antibodies) are those whoseinteractions with antigen change with calcium concentration. Acalcium-dependent antigen-binding antibody is thought to bind to anantigen through calcium ion. Thus, amino acids that form an epitope onthe antigen side are negatively charged amino acids that can chelatecalcium ions or amino acids that can be a hydrogen-bond acceptor. Theseproperties of amino acids that form an epitope allows targeting of anepitope other than antigen-binding molecules, which are generated byintroducing histidines and bind to an antigen in a pH-dependent manner.Furthermore, the use of antigen-binding molecules having calcium- andpH-dependent antigen-binding properties is thought to allow theformation of antigen-binding molecules that can individually targetvarious epitopes having broad properties. Thus, if a population ofmolecules containing a calcium-binding motif (Ca library) isconstructed, from which antigen-binding molecules are obtained,calcium-dependent antigen-binding molecules are thought to beeffectively obtained.

(33-2) Acquisition of Human Germline Sequences

An example of the population of molecules containing a calcium-bindingmotif is an example in which said molecules are antibodies. In otherwords, an antibody library containing a calcium-binding motif may be aCa library.

Calcium ion-binding antibodies containing human germline sequences havenot been reported. Thus, each antibody having human germline sequenceswere cloned using as a template cDNA prepared from Human Fetal SpleenPoly RNA (Clontech) to assess whether antibodies having human germlinesequences bind to calcium ion. Cloned DNA fragments were inserted intoanimal cell expression vectors. The nucleotide sequences of theconstructed expression vectors were determined by a method known tothose skilled in the art. The SEQ IDs are shown in Table 45. By PCR,polynucleotides encoding SEQ ID NO: 58 (Vk1), SEQ ID NO: 59 (Vk2), SEQID NO: 60 (Vk3), SEQ ID NO: 61 (Vk4), and SEQ ID NO: 62 (Vk5-2) werelinked to a polynucleotide encoding the natural Kappa chain constantregion (SEQ ID NO: 54). The linked DNA fragments were inserted intoanimal cell expression vectors. Furthermore, heavy chain variable regionpolynucleotides encoding SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,SEQ ID NO: 66, and SEQ ID NO: 67 were linked by PCR to a polynucleotideencoding an IgG1 of SEQ ID NO: 53 (having a deletion of two amino acidsat the C terminus of natural sequence). The resulting DNA fragments wereinserted into animal cell expression vectors. The sequences of theconstructed variants were confirmed by a method known to those skilledin the art.

TABLE 45 LIGHT CHAIN HEAVY CHAIN LIGHT CHAIN GERMLINE (VARIABLE REGION)VARIABLE REGION SEQUENCE SEQ ID NO SEQ ID NO Vk1 63 58 Vk2 64 59 Vk3 6560 Vk4 66 61 Vk5 67 62

(33-3) Expression and Purification of Antibodies

The constructed animal cell expression vectors inserted with the DNAfragments having the five types of human germline sequences wereintroduced into animal cells. Antibody expression was carried out by thefollowing method. Cells of human fetal kidney cell-derived FreeStyle293-F (Invitrogen) were suspended in the FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)into each well of a 6-well plate. The prepared plasmids are introducedinto cells by a lipofection method. The cells were cultured for fourdays in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). From the culturesupernatants prepared as described above, antibodies were purified usingthe rProtein A Sepharose™ Fast Flow (Amersham Biosciences) by a methodknown to those skilled in the art. Absorbance at 280 nm of the purifiedantibody solutions was measured using a spectrophotometer. Antibodyconcentrations were calculated from the determined values using anextinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

(33-4) Assessment of Antibodies Having Human Germline Sequences fortheir Calcium Ion-Binding Activity

The purified antibodies were assessed for their calcium ion-bindingactivity. The intermediate temperature of thermal denaturation (Tmvalue) was measured by differential scanning calorimetry (DSC) as anindicator for examining calcium ion binding to the antibody (MicroCalVP-Capillary DSC, MicroCal). The intermediate temperature of thermaldenaturation (Tm value) is an indicator of stability. It becomes higherwhen a protein is stabilized through calcium ion binding, as comparedwith the case where no calcium ion is bound (J. Biol. Chem. (2008) 283,37, 25140-25149). The binding activity of calcium ion to antibody wasevaluated by examining changes in the Tm value of the antibody dependingon the changes in the calcium ion concentration in the antibodysolution. The purified antibody was subjected to dialysis (EasySEP,TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2mM CaCl₂ (pH 7.4) or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl₂ (pH7.4). DSC measurement was conducted at a heating rate of 240° C./hr from20 to 115° C. using as a test substance an antibody solution prepared atabout 0.1 mg/mL with the dialysate. The intermediate temperatures ofthermal denaturation (Tm values) of the Fab domains of each antibody,calculated from the denaturation curve obtained by DSC, are shown inTable 46.

TABLE 46 CALCIUM ION LIGHT CHAIN CONCENTRATION ΔTm (° C.) GERMLINESEQUENCE 3 μM 2 mM 2 mM − 3 μM hVk1 80.32 80.78 0.46 hVk2 80.67 80.61−0.06 hVk3 81.64 81.36 −0.28 hVk4 70.74 70.74 0 hVk5 71.52 74.17 2.65

The result showed that the Tm values of the Fab domains of antibodieshaving the hVk1, hVk2, hVk3, or hVk4 sequence did not vary depending onthe calcium ion concentration in the Fab domain-containing solutions.Meanwhile, the Tm value for the antibody Fab domain having the hVk5sequence varied depending on the calcium ion concentration in the Fabdomain-containing solution. This demonstrates that the hVk5 sequencebinds to calcium ion.

(33-5) Assessment of hVk5-2 Sequence for Calcium Binding

In (33-2), Vk5-2 variant 1 (SEQ ID NO: 68) and Vk5-2 variant 2 (SEQ IDNO: 69) were obtained in addition to Vk5-2 (SEQ ID NO: 57), all of whichare classified as Vk5-2. These variants were assessed for their calciumbinding. DNA fragments for Vk5-2, Vk5-2 variant 1, and Vk5-2 variant 2were each inserted into animal cell expression vectors. The nucleotidesequences of the constructed expression vectors were determined by amethod known to those skilled in the art. By the method described in(33-3), the animal cell expression vectors inserted with DNA fragmentsfor Vk5-2, Vk5-2 variant 1, and Vk5-2 variant 2 were introduced, incombination with animal expression vector carrying an insert to expressCIM_H (SEQ ID NO: 67) as a heavy chain, into animal cells and antibodieswere purified. The purified antibodies were assessed for their calciumion-binding activity. The purified antibodies were dialyzed (EasySEP,TOMY) against 20 mM Tris-HCl/150 mM NaCl (pH 7.5) (in Table 47,indicated as 0 mM calcium ion concentration) or 20 mM Tris-HCl/150 mMNaCl/2 mM CaCl₂ (pH 7.5). DSC measurement was carried out at a rate oftemperature increase of 240° C./hr from 20 to 115° C. using as a testsubstance an antibody solution prepared at a concentration of about 0.1mg/mL with the same solution as used for dialysis. Based on the obtainedDSC denaturation curve, the intermediate temperature of thermaldenaturation (Tm value) was calculated for the Fab domain of eachantibody. The Tm values are shown in Table 47.

TABLE 47 CALCIUM ION CONCENTRATION ΔTm (° C.) LIGHT CHAIN 0 mM 2 mM 2 mM− 0 mM Vk5-2 71.65 74.38 2.73 Vk5-2 VARIANT 1 65.75 72.24 6.49 Vk5-2VARIANT 2 66.46 72.24 5.78

The result showed that the Tm value for the Fab domains of antibodieshaving the sequence of Vk5-2, Vk5-2 variant 1, or Vk5-2 variant 2 varieddepending on the calcium ion concentration in solutions containingantibodies having the Fab domains. This demonstrates that antibodieshaving a sequence classified as Vk5-2 bind to calcium ion.

Reference Example 34 Assessment of the Human Vk5 (hVk5) Sequence

(34-1) hVk5 Sequence

The only hVk5 sequence registered in Kabat database is hVk5-2 sequence.Herein, hVk5 and hVk5-2 are used synonymously. WO2010/136598 disclosesthat the abundance ratio of the hVk5-2 sequence in the germline sequenceis 0.4%. Other reports have been also made in which the abundance ratioof the hVk5-2 sequence in the germline sequence is 0-0.06% (J. Mol.Biol. (2000) 296, 57-86; Proc. Natl. Acad. Sci. USA (2009) 106, 48,20216-20221). As described above, since the hVk5-2 sequence is asequence of low appearance frequency in the germline sequence, it wasthought to be inefficient to obtain a calcium-binding antibody from anantibody library consisting of human germline sequences or B cellsobtained by immunizing a mouse expressing human antibodies. Thus, it ispossible to design Ca libraries containing the sequence of human hVk5-2.Meanwhile, reported synthetic antibody libraries (WO2010/105256 andWO2010/136598) did not contain the sequence of hVk5. In addition,realization of the possibility is unknown because no report has beenpublished on the physical properties of the hVk5-2 sequence.

(34-2) Construction, Expression, and Purification of a Non-GlycosylatedForm of the hVk5-2 Sequence

The hVk5-2 sequence has a sequence for N-type glycosylation at position20 amino acid (Kabat numbering). Sugar chains attached to proteinsexhibit heterogeneity. Thus, it is desirable to lose the glycosylationfrom the viewpoint of substance homogeneity. In this context, varianthVk5-2_L65 (SEQ ID NO: 70) in which the Asn (N) residue at position 20(Kabat numbering) is substituted with Thr (T) was constructed. Aminoacid substitution was carried out by a method known to those skilled inthe art using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).A DNA encoding the variant hVk5-2_L65 was inserted into an animalexpression vector. The animal expression vector inserted with theconstructed DNA encoding variant hVk5-2_L65, in combination with ananimal expression vector having an insert to express CIM_H (SEQ ID NO:67) as a heavy chain, was introduced into animal cells by the methoddescribed in Reference Example 25. The antibody comprising hVk5-2_L65and CIM_H, which was expressed in animal cells introduced with thevectors, was purified by the method described in Reference Example 33.

(34-3) Assessment of the Antibody Having the Non-Glycosylated hVk5-2Sequence for Physical Properties

The isolated antibody having the modified sequence hVk5-2_L65 wasanalyzed by ion-exchange chromatography to test whether it is lessheterogeneous than the antibody having the original sequence hVk5-2before alteration. The procedure of ion-exchange chromatography is shownin Table 48. The analysis result showed that hVk5-2_L65 modified at theglycosylation site was less heterogeneous than the original sequencehVk5-2, as shown in FIG. 57.

TABLE 48 CONDITION COLUMN TOSOH TSKgel DEAE-NPR MOBILE PHASE A; 10 mMTris-HCl, 3 μM CaCl₂ (pH 8.0) B; 10 mM Tris-HCl, 500 mM NaCl, 3 μM CaCl₂(pH 8.0) GRADIENT % B = 0 − (5 min) − 0 − 2%/1 min SCHEDULE COLUMN 40°C. TEMPERATURE DETECTION 280 nm INJECTION 100 μL (5 μg) VOLUME

Next, whether the less-heterogeneous hVk5-2_L65 sequence-comprisingantibody binds to calcium ion was assessed by the method described inReference Example 33. The result showed that the Tm value for the Fabdomain of the antibody having hVk5-2_L65 with altered glycosylation sitealso varied depending on the calcium ion concentration in the antibodysolutions, as shown in Table 49. Specifically, it was demonstrated thatthe Fab domain of the antibody having hVk5-2_L65 with alteredglycosylation site binds to calcium ion.

TABLE 49 GLYCO- CALCIUM ION LIGHT SYLATED CONCENTRATION ΔTm (° C.) CHAINSEQUENCE 3 μM 2 mM 2 mM − 3 μM hVk5-2 YES 71.52 74.17 2.65 hVk5-2_L65 NO71.51 73.66 2.15

Reference Example 35 Assessment of the Calcium Ion-Binding Activity ofAntibody Molecules Having CDR Sequence of the hVk5-2 Sequence

(35-1) Construction, Expression, and Purification of Modified AntibodiesHaving a CDR Sequence from the hVk5-2 Sequence

The hVk5-2_L65 sequence is a sequence with altered amino acids at aglycosylation site in the framework of human Vk5-2 sequence. Asdescribed in Reference Example 34, it was demonstrated that calcium ionbound even after alteration of the glycosylation site. Meanwhile, fromthe viewpoint of immunogenicity, it is generally desirable that theframework sequence is a germline sequence. Thus, the present inventorsassessed whether an antibody framework sequence could be substitutedwith the framework sequence of a non-glycosylated germline sequencewhile maintaining the calcium ion-binding activity of the antibody.

Polynucleotides encoding chemically synthesized sequences in whichframework sequence of the hVk5-2 sequence is altered with hVk1, hVk2,hVk3, or hVk4 (CaVk1 (SEQ ID NO: 71), CaVk2 (SEQ ID NO: 72), CaVk3 (SEQID NO: 73), or CaVk4 (SEQ ID NO: 74), respectively) were linked by PCRto a polynucleotide encoding the constant region (SEQ ID NO: 54) of thenatural Kappa chain. The linked DNA fragments were inserted into animalcell expression vectors. Sequences of the constructed variants wereconfirmed by a method known to those skilled in the art. Each plasmidconstructed as described above was introduced into animal cells incombination with a plasmid inserted with a polynucleotide encoding CIM_H(SEQ ID NO: 67) by the method described in Reference Example 33. Theexpressed antibody molecules of interest were purified from culturemedia of the animal cells introduced with the plasmids.

(35-2) Assessment of Altered Antibodies Having the CDR Sequence of thehVk5-2 Sequence for their Calcium Ion-Binding Activity

Whether calcium ion binds to altered antibodies having the CDR sequenceof the hVK5-2 sequence and the framework sequences of germline sequencesother than hVk5-2 (hVk1, hVk2, hVk3, and hVk4) was assessed by themethod described in Reference Example 25. The assessment result is shownin Table 50. The Tm value of the Fab domain of each altered antibody wasrevealed to vary depending on the calcium ion concentration in theantibody solutions. This demonstrates that antibodies having a frameworksequence other than the hVk5-2 sequence also bind to calcium ion.

TABLE 50 GERMLINE (LIGHT CHAIN CALCIUM ION FRAMEWORK CONCENTRATION ΔTm(° C.) SEQUENCE) 3 μM 2 mM 2 mM − 3 μM hVk1 77.51 79.79 2.28 hVk2 78.4680.37 1.91 hVk3 77.27 79.54 2.27 hVk4 80.35 81.38 1.03 hVk5-2 71.5274.17 2.65

The thermal denaturation temperature (Tm value), as an indicator ofthermal stability, of the Fab domain of each antibody altered to havethe CDR sequence of the hVK5-2 sequence and the framework sequence of agermline sequence other than the hVk5-2 sequence (hVk1, hVk2, hVk3, orhVk4) was demonstrated to be greater than that of the Fab domain of theoriginal antibody having the hVk5-2 sequence. This result shows thatantibodies having the CDR sequence of the hVk5-2 sequence and theframework sequence of hVk1, hVk2, hVk3, or hVk4 not only have calciumion-binding activity but also are excellent molecules from the viewpointof thermal stability.

Reference Example 36 Identification of the Calcium Ion-Binding Site inHuman Germline hVk5-2 Sequence

(36-1) Design of Mutation Site in the CDR Sequence of the hVk5-2Sequence

As described in Reference Example 35, antibodies having the light chainresulting from introduction of the CDR sequence of the hVk5-2 sequenceinto the framework sequence of a different germline sequence were alsodemonstrated to bind to calcium ion. This result suggests that inhVk5-2a calcium ion-binding site is localized within its CDR sequence.Amino acids that bind to calcium ion, i.e., chelate calcium ion, includenegatively charged amino acids and amino acids that can be a hydrogenbond acceptor. Thus, it was tested whether antibodies having a mutanthVk5-2 sequence with a substitution of an Ala (A) residue for an Asp (D)or Glu (E) residue in the CDR sequence of the hVk5-2 sequence bind tocalcium ion.

(36-2) Construction of Variant hVk5-2 Sequences with Ala Substitution,and Expression and Purification of Antibodies

Antibody molecules were prepared to comprise a light chain withsubstitution of an Ala residue for Asp and/or Glu residue in the CDRsequence of the hVk5-2 sequence. As described in Reference Example 34,non-glycosylated variant hVk5-2_L65 exhibited calcium ion binding andwas assumed to be equivalent to the hVk5-2 sequence in terms of calciumion binding. In this Example, amino acid substitutions were introducedinto hVk5-2_L65 as a template sequence. Constructed variants are shownin Table 51. Amino acid substitutions were carried out by methods knownto those skilled in the art such as using the QuikChange Site-DirectedMutagenesis Kit (Stratagene), PCR, or the In fusion Advantage PCRCloning Kit (TAKARA) to construct expression vectors for altered lightchains having an amino acid substitution.

TABLE 51 LIGHT CHAIN ALTERED POSITION VARIANT NAME (Kabat NUMBERING) SEQID NO hVk5-2_L65 WILDTYPE 70 hVk5-2_L66 30 75 hVk5-2_L67 31 76hVk5-2_L68 32 77 hVk5-2_L69 50 78 hVk5-2_L70 30, 32 79 hVk5-2_L71 30, 5080 hVk5-2_L72 30, 32, 50 81 hVk5-2_L73 92 82

Nucleotide sequences of the constructed expression vectors wereconfirmed by a method known to those skilled in the art. The expressionvectors constructed for the altered light chains were transientlyintroduced, in combination with an expression vector for the heavy chainCIM_H (SEQ ID NO: 67), into cells of the human fetal kidney cell-derivedHEK293H line (Invitrogen) or FreeStyle293 (Invitrogen) to expressantibodies. From the obtained culture supernatants, antibodies werepurified using the rProtein A Sepharose™ Fast Flow (GE Healthcare) by amethod known to those skilled in the art. Absorbance at 280 nm of thepurified antibody solutions was measured using a spectrophotometer.Antibody concentrations were calculated from the determined values usingan extinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

(36-3) Assessment of the Calcium Ion-Binding Activity of AntibodiesHaving an Ala Substitution in the hVk5-2 Sequence

Whether the obtained purified antibodies bind to calcium ion was testedby the method described in Reference Example 33. The result is shown inTable 52. Some antibodies having substitution of an Asp or Glu residuein the CDR sequence of the hVk5-2 sequence with an Ala residue whichcannot be involved in calcium ion binding or chelation were revealed tohave an Fab domain whose Tm did not vary by the calcium ionconcentration in the antibody solutions. The substitution sites at whichAla substitution did not alter the Tm (positions 32 and 92 (Kabatnumbering)) were demonstrated to be greatly important for the calciumion-antibody binding.

TABLE 52 LIGHT ALTERED CHAIN POSITION CALCIUM ION VARIANT (KabatCONCENTRATION ΔTm (° C.) NAME NUMBERING) 0 μM 2 mM 2 mM − 0 μMhVk5-2_L65 WILDTYPE 71.71 73.69 1.98 hVk5-2_L66 30 71.65 72.83 1.18hVk5-2_L67 31 71.52 73.30 1.78 hVk5-2_L68 32 73.25 74.03 0.78 hVk5-2_L6950 72.00 73.97 1.97 hVk5-2_L70 30, 32 73.42 73.60 0.18 hVk5-2_L71 30, 5071.84 72.57 0.73 hVk5-2_L72 30, 32, 50 75.04 75.17 0.13 hVk5-2_L73 9275.23 75.04 −0.19

Reference Example 37 Assessment of the Antibodies Having hVk1 Sequencewith Calcium Ion-Binding Motif

(37-1) Construction of an hVk1 Sequence with Calcium Ion-Binding Motif,and Expression and Purification of Antibodies

The result described in Reference Example 36 on the calcium-bindingactivity of the Ala substitute demonstrates that Asp or Glu residues inthe CDR sequence of the hVk5-2 sequence were important for calciumbinding. Thus, the present inventors assessed whether an antibody canbind to calcium ion when the residues at positions 30, 31, 32, 50, and92 (Kabat numbering) alone were introduced into a different germlinevariable region sequence. Specifically, variant LfVk1_Ca (SEQ ID NO: 83)was constructed by substituting the residues at positions 30, 31, 32,50, and 92 (Kabat numbering) in the hVk5-2 sequence for the residues atpositions 30, 31, 32, 50, and 92 (Kabat numbering) in the hVk1 sequence(a human germline sequence). Specifically, it was tested whetherantibodies having an hVk1 sequence introduced with only 5 residues fromthe hVk5-2 sequence can bind to calcium. The variants were produced bythe same method as described in Reference Example 36. The resultinglight chain variant LfVk1_Ca and LfVk1 having the light-chain hVk1sequence (SEQ ID NO: 84) were co-expressed with the heavy chain CIM_H(SEQ ID NO: 67). Antibodies were expressed and purified by the samemethod as described in Reference Example 36.

(37-2) Assessment of the Calcium Ion-Binding Activity of AntibodiesHaving a Human hVk1 Sequence with Calcium Ion-Binding Motif

Whether the purified antibody prepared as described above binds tocalcium ion was assessed by the method described in Reference Example33. The result is shown in Table 53. The Tm value of the Fab domain ofthe antibody having LfVk1 with an hVk1 sequence did not vary dependingon the calcium concentration in the antibody solutions. Meanwhile, Tm ofthe antibody having the LfVk1_Ca sequence was shifted by 1° C. or moreupon change in the calcium concentration in the antibody solutions.Thus, it was shown that the antibody having LfVk1_Ca binds to calcium.The result described above demonstrates that the entire CDR sequence ofhVk5-2 is not required, while the residues introduced for constructionof the LfVk1_Ca sequence alone are sufficient for calcium ion binding.

TABLE 53 CALCIUM ION LIGHT CHAIN CONCENTRATION ΔTm (° C.) VARIANT 3 μM 2mM 2 mM − 3 μM LfVk1 83.18 83.81 0.63 LfVk1_Ca 79.83 82.24 2.41

(37-3) Construction, Expression, and Purification ofDegradation-Resistant LfVk1_Ca Sequence

As described in (37-1), variant LfVk1_Ca (SEQ ID NO: 83) was constructedto have substitution of residues at positions 30, 31, 32, 50, and 92(Kabat numbering) in the hVk5-2 sequence for residues at positions 30,31, 32, 50, and 92 (Kabat numbering) in the hVk1 sequence (a humangermline sequence). The variant was demonstrated to bind to calcium ion.Thus, it is possible to design Ca libraries containing LfVk1_Casequence. Meanwhile, there is no report on the properties of LfVk1_Casequence, and thus its feasibility was unknown. LfVk1_Ca sequence hasAsp at positions 30, 31, and 32 (Kabat numbering). Thus, the Asp-Aspsequence which has been reported to be degraded under acidic conditionis contained in the CDR1 sequence (J. Pharm. Biomed. Anal. (2008) 47(1),23-30). It is desirable to avoid the degradation at acidic conditionsfrom the viewpoint of the storage stability of antibody. Then, variantsLfVk1_Ca1 (SEQ ID NO: 85), LfVk1_Ca2 (SEQ ID NO: 86), and LfVk1_Ca3 (SEQID NO: 87) were constructed to have substitution of Ala (A) residues forAsp (D) residues that are possibly sensitive to degradation. Amino acidsubstitution was carried out by a method known to those skilled in theart using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).DNAs encoding the variants were inserted into animal expression vectors.In combination with an animal expression vector having an insert toexpress GC_H (SEQ ID NO: 55) as the heavy chain, the constructed animalexpression vectors carrying DNA inserts for the variants were introducedinto animal cells by the method described in Reference Example 33. Theantibodies expressed in the animal cells introduced with the vectorswere purified by the method described in Reference Example 33.

(37-4) Stability Assessment of Antibodies Having theDegradation-Resistant LfVk1_Ca Sequence

Whether the antibodies prepared as described in (37-3) were moreresistant to degradation in solutions at pH 6.0 than the originalantibodies having the LfVk1_Ca sequence provided for alteration wasassessed by comparing the heterogeneity between respective antibodiesafter thermal acceleration. Each antibody was dialyzed against asolution of 20 mM Histidine-HCl, 150 mM NaCl (pH 6.0) under a conditionof 4° C. overnight. Dialyzed antibodies were adjusted to 0.5 mg/mL andstored at 5° C. or 50° C. for three days. Each antibody after storagewas subjected to ion-exchange chromatography using the method describedin Reference Example 34. As shown in FIG. 58, the analysis resultdemonstrates that LfVk1_Ca1 with an alteration at degradation site wasless heterogeneous and much more resistant to degradation from thermalacceleration than the original LfVk1_Ca sequence. Specifically, it wasdemonstrated that degradation occurred at the Asp (D) residue ofposition 30 in the LfVk1_Ca sequence but it could be prevented by aminoacid alteration.

(37-5) Construction of a Light Chain LVk1_Ca Sequence Resistant toDegradation at the Asp Residue of Position 30, and Expression andPurification of Antibodies

The result described in (37-4) on the degradation resistance of theAla-substituted form demonstrates that under acidic conditions theLfVk1_Ca sequence was degraded at the Asp (D) residue of position 30(Kabat numbering) in its CDR sequence and the degradation could beprevented in the case of substitution of a different amino acid (in(37-4), an Ala (A) residue) for the Asp (D) residue at position 30(Kabat numbering). Then, the present inventors tested whether even asequence with a substitution of Ser (S), a main residue capable ofchelating calcium ion, for the residue at position 30 (Kabat numbering)(referred to as LfVk1_Ca6; SEQ ID NO: 88) was resistant to degradation.Variants were prepared by the same method as described in ReferenceExample 29. The altered light chains LfVk1_Ca6 and LfVk1_Ca sequenceswere expressed in combination with a heavy chain GC_H (SEQ ID NO: 55).Antibodies were expressed and purified by the same method as describedin Reference Example 36.

(37-6) Assessment of a Light Chain LVk1_Ca Sequence Resistant toDegradation at Asp Residue at Position 30

Purified antibodies prepared as described above were assessed for theirstorage stability under acidic conditions by the method described in(37-4). The result demonstrates that antibodies having the LfVk1_Ca6sequence are more resistant to degradation than those having theoriginal LfVk1_Ca sequence, as shown in FIG. 59.

Then, whether antibodies having the LfVk1_Ca sequence and antibodieshaving the LfVk1_Ca6 sequence bind to calcium ion was tested by themethod described in Reference Example 33. The result is shown in Table54. The Tm values of the Fab domains of antibodies having LfVk1_Casequence and antibodies having the degradation-resistant LfVk1_Ca6sequence were shifted by 1° C. or more upon change in the calciumconcentration in antibody solutions.

TABLE 54 CALCIUM ION LIGHT CHAIN CONCENTRATION ΔTm (° C.) VARIANT 3 μM 2mM 2 mM − 3 μM LfVk1_Ca 78.45 80.06 1.61 LfVk1_Ca6 78.44 79.74 1.30

Reference Example 38 Design of a Population of Antibody Molecules (CaLibrary) with a Calcium Ion-Binding Motif Introduced into the VariableRegion to Effectively Obtain Antibodies that Bind to Antigen in a CaConcentration-Dependent Manner

Preferred calcium-binding motifs include, for example, the hVk5-2sequence and its CDR sequence, as well as residues at positions 30, 31,32, 50, and 92 (Kabat numbering) thereof. Other calcium binding motifsinclude the EF-hand motif possessed by calcium-binding proteins (e.g.,calmodulin) and C-type lectin (e.g., ASGPR).

The Ca library consists of heavy and light chain variable regions. Ahuman antibody sequence was used for the heavy chain variable region,and a calcium-binding motif was introduced into the light chain variableregion. The hVk1 sequence was selected as a template sequence of thelight chain variable region for introducing a calcium-binding motif. Anantibody containing an LfVk1_Ca sequence obtained by introducing the CDRsequence of hVk5-2 (one of calcium-binding motifs) into the hVk1sequence was shown to bind to calcium ions, as shown in ReferenceExample 36. Multiple amino acids were allowed to appear in the templatesequence to diversify antigen-binding molecules that constitute thelibrary. Positions exposed on the surface of a variable region which islikely to interact with the antigen were selected as those wheremultiple amino acids are allowed to appear. Specifically, positions 30,31, 32, 34, 50, 53, 91, 92, 93, 94, and 96 (Kabat numbering) wereselected as flexible residues.

The type and appearance frequency of amino acid residues that weresubsequently allowed to appear were determined. The appearance frequencyof amino acids in the flexible residues of the hVk1 and hVk3 sequencesregistered in the Kabat database (KABAT, E. A. ET AL.: ‘Sequences ofproteins of immunological interest’, vol. 91, 1991, NIH PUBLICATION) wasanalyzed. Based on the analysis results, the type of amino acids thatwere allowed to appear in the Ca library were selected from those withhigher appearance frequency at each position. At this time, amino acidswhose appearance frequency was determined to be low based on theanalysis results were also selected to avoid the bias of amino acidproperties. The appearance frequency of the selected amino acids wasdetermined in reference to the analysis results of the Kabat database.

A Ca library containing a calcium-binding motif with emphasis on thesequence diversity as to contain multiple amino acids at each residueother than the motif were designed as a Ca library in consideration ofthe amino acids and appearance frequency set as described above. Thedetailed designs of the Ca library are shown in Tables 9 and 10 (withthe positions in each table representing the Kabat numbering). Inaddition, in Tables 9 and 10, if position 92 represented by the Kabatnumbering is Asn (N), position 94 may be Leu (L) instead of Ser (S).

Reference Example 39 Ca Library Preparation

A gene library of antibody heavy-chain variable regions was amplified byPCR using a poly A RNA prepared from human PBMC, and commercial humanpoly A RNA, etc. as a template. As described in Reference Example 38,for the light chain variable regions of antibody, light chain variableregions of antibody that increase appearance frequency of antibodieswhich maintain a calcium-binding motif and can bind to an antigen in acalcium concentration-dependent manner were designed. In addition, foramino acid residues other than those with a calcium-binding motifintroduced, a library of antibody light chain variable regions withevenly distributed amino acids of high appearance frequency in naturalhuman antibodies as flexible residues was designed with reference to theinformation of amino acid appearance frequency in natural humanantibodies (KABAT, E. A. ET AL.: ‘Sequences of proteins of immunologicalinterest’, vol. 91, 1991, NIH PUBLICATION). A combination of the genelibraries of antibody heavy-chain and light-chain variable regionsgenerated as described above, was inserted into a phagemid vector toconstruct a human antibody phage display library that presents Fabdomains consisting of human antibody sequences (Methods Mol. Biol.(2002) 178, 87-100).

The sequences of antibody genes isolated from E. coli introduced with anantibody gene library were determined according to the method describedin Reference Example 43 below. The amino acid distribution in thesequences of isolated 290 clones and a designed amino acid distributionare shown in FIG. 60.

Reference Example 40 Examination of the Calcium Ion-Binding Activity ofMolecules Contained in the Ca Library (40-1) Calcium Ion-BindingActivity of Molecules Contained in the Ca Library

As described in Reference Example 34, the hVk5-2 sequence that wasdemonstrated to bind to calcium ions is a sequence of low appearancefrequency in the germline sequence. Thus, it was thought to beinefficient to obtain a calcium-binding antibody from an antibodylibrary consisting of human germline sequences or from B cells obtainedby immunizing a mouse expressing human antibodies. As a result, a Calibrary was constructed. The presence or absence of a clone showingcalcium binding in the constructed Ca library was examined.

(40-2) Expression and Purification of Antibodies

Clones contained in the Ca library were introduced into animal cellexpression plasmids. Antibodies were expressed using the methoddescribed below. Cells of human fetal kidney cell-derived FreeStyle293-F line (Invitrogen) were suspended in FreeStyle 293 ExpressionMedium (Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml(3 ml) to each well of a 6-well plate. The prepared plasmids wereintroduced into the cells by a lipofection method. The cells werecultured in a CO₂ incubator (37° C., 8% CO₂, 90 rpm) for four days. By amethod known to those skilled in the art, antibodies were purified usingrProtein A Sepharose™ Fast Flow (Amersham Biosciences) from culturesupernatants obtained as described above. The absorbance of solutions ofpurified antibodies was measured at 280 nm using a spectrophotometer.Antibody concentrations were calculated from the measured values byusing the extinction coefficient determined by PACE method (ProteinScience (1995) 4, 2411-2423).

(40-3) Assessment of Prepared Antibodies for their Calcium Ion Binding

Antibodies purified as described above were assessed for their calciumion binding by the method described in Reference Example 26. The resultis shown in Table 55. The Tm of the Fab domains of multiple antibodiescontained in the Ca library changed depending on calcium ionconcentration, suggesting that the library contains molecules that bindto calcium ion.

TABLE 55 SEQ ID NO CALCIUM ION ANTI- HEAVY LIGHT CONCENTRATION ΔTm (°C.) BODY CHAIN CHAIN 3 μM 2 mM 2 mM − 3 μM Ca_B01 89 100 70.88 71.450.57 Ca_E01 90 101 84.31 84.95 0.64 Ca_H01 91 102 77.87 79.49 1.62Ca_D02 92 103 78.94 81.1 2.16 Ca_E02 93 104 81.41 83.18 1.77 Ca_H02 94105 72.84 75.13 2.29 Ca_D03 95 106 87.39 86.78 −0.61 Ca_C01 96 107 74.7474.92 0.18 Ca_G01 97 108 65.21 65.87 0.66 Ca_A03 98 109 80.64 81.89 1.25Ca_B03 99 110 93.02 93.75 0.73

Reference Example 41 Isolation of Antibodies that Bind to IL-6 Receptorin a Ca-Dependent Manner

(41-1) Isolation of Antibody Fragments, which Bind to Antigens in aCa-Dependent Manner, from Library by Bead Panning

The first selection from the constructed library of antibodies that bindin a Ca-dependent manner was performed by enriching only antibodyfragments having the ability to bind to the antigen (IL-6 receptor).

Phages were produced by E. coli containing the constructed phagemids forphage display. To precipitate the phages, 2.5 M NaCl/10% PEG was addedto the E. coli culture media of phage production. The precipitated phagepopulation was diluted with TBS to prepare a phage library solution.Then, BSA and CaCl₂ were added to the phage library solution to adjustthe final BSA concentration to 4% and the final calcium ionconcentration to 1.2 mM. Regarding the panning method, the presentinventors referred to general panning methods using antigens immobilizedonto magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J.Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002)18(2) 212-20; Mol. Cell. Proteomics (2003) 2 (2), 61-9). The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to theprepared phage library solution to allow the contact of the phagelibrary solution with the antigen at room temperature for 60 minutes.BSA-blocked magnetic beads were added and allowed to bind toantigen/phage complexes at room temperature for 15 minutes. The beadswere washed three times with 1 ml of 1.2 mM CaCl₂/TBST (TBST containing1.2 mM CaCl₂) and then twice with 1 ml of 1.2 mM CaCl₂/TBS (TBSTcontaining 1.2 mM CaCl₂). Then, the beads combined with 0.5 ml of 1mg/ml trypsin were suspended at room temperature for 15 minutes, andimmediately followed by separation of beads using a magnetic stand tocollect a phage solution. The collected phage solution was added to 10ml of E. coli strain ER2738 in a logarithmic growth phase (OD600 of0.4-0.7). The E. coli was infected with the phages by culturing themwhile gently stirring at 37° C. for one hour. The infected E. coli wasplated in a 225 mm×225 mm plate. Then, the phages were collected fromthe culture medium of the plated E. coli to prepare a phage librarysolution.

In the second-round panning, phages were enriched using theantigen-binding ability or the Ca-dependent binding ability as anindicator.

Specifically, when the enrichment was carried out using theantigen-binding ability as an indicator, 40 pmol of biotin-labeledantigen was added to the prepared phage library solution to allow thecontact of the phage library solution with the antigen at roomtemperature for 60 minutes. BSA-blocked magnetic beads were added andallowed to bind to antigen/phage complexes at room temperature for 15minutes. The beads were washed three times with 1 ml of 1.2 mMCaCl₂/TBST and then twice with 1.2 mM CaCl₂/TBS. Then, the beads addedwith 0.5 ml of 1 mg/ml trypsin were suspended at room temperature for 15minutes. Then immediately, the beads were separated using a magneticstand to collect a phage solution. To eliminate the ability from phagesdisplaying no Fab to infect E. coli, the pIII protein (helperphage-derived pIII protein) of phages displaying no Fab was cleaved byadding 5 μl of 100 mg/ml trypsin to the collected phage solution. Therecovered phage solution was added to 10 mL of the E. coli strain ER2738in a logarithmic growth phase (OD600 of 0.4-0.7). The E. coli wascultured with gentle stirring at 37° C. for 1 hour to allow the phagesto infect the E. coli. The infected E. coli was inoculated into a 225mm×225 mm plate. Subsequently, the phages were recovered from theculture medium of the E. coli after inoculation to collect a phagelibrary solution.

When the enrichment was carried out using the Ca-dependent bindingability as an indicator, 40 pmol of biotin-labeled antigen was added tothe prepared phage library solution to allow the contact of the phagelibrary solution with the antigen at room temperature for 60 minutes.BSA-blocked magnetic beads were added and allowed to bind toantigen/phage complexes at room temperature for 15 minutes. The beadswere washed with 1 ml of 1.2 mM CaCl₂/TBST and 1.2 mM CaCl₂/TBS. Then,the beads added with 0.1 ml of 2 mM EDTA/TBS (TBS containing 2 mM EDTA)were suspended at room temperature. Then immediately, the beads wereseparated using a magnetic stand to collect a phage solution. Toeliminate the ability from phages displaying no Fab to infect E. coli,the pIII protein (helper phage-derived pIII protein) of phagesdisplaying no Fab was cleaved by adding 5 μl of 100 mg/ml trypsin to thecollected phage solution. The recovered phage solution was added to 10mL of the E. coli strain ER2738 in a logarithmic growth phase (OD600 of0.4-0.7). The E. coli was cultured with gentle stirring at 37° C. for 1hour to allow the phages to infect the E. coli. The infected E. coli wasinoculated into a 225 mm×225 mm plate. Subsequently, the phages wererecovered from the culture medium of the E. coli after inoculation tocollect a phage library solution.

(41-2) Examination by Phage ELISA

A phage-containing culture supernatant was collected according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from a singlecolony of E. coli, obtained as described above.

A culture supernatant containing phages, to which BSA and CaCl₂ wereadded was subjected to ELISA as described below. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μL of PBScontaining the biotin-labeled antigen. Each well of said plate waswashed with PBST to remove the antigen, and then the wells were blockedwith 250 μL of 4% BSA-TBS for 1 hour or longer. Said plate with theprepared culture supernatant added to each well, from which the 4%BSA-TBS was removed, was allowed to stand undisturbed at 37° C. for 1hour, allowing the binding of phage-presenting antibody to the antigenpresent in each well. To each well washed with 1.2 mM CaCl₂/TBST, 1.2 mMCaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was allowed to standundisturbed for 30 minutes at 37° C. for incubation. After washing with1.2 mM CaCl₂/TBST, an HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS at a concentration of 1.2 mM ofionized calcium concentration was added to each well, and the plate wasincubated for 1 hour. After washing with 1.2 mM CaCl₂/TBST, thechromogenic reaction of the solution in each well with a TMB singlesolution (ZYMED) added was stopped by adding sulfuric acid.Subsequently, said developed color was measured by measuring absorbanceat 450 nm.

The base sequences of genes amplified with specific primers wereanalyzed for the clones subjected to phage ELISA.

The result of phage ELISA and sequence analysis is shown in Table 56.

TABLE 56 LIBRARY Ca LIBRARY Ca LIBRARY ENRICHMENT INDEX ANTIGEN-BINDINGDEPENDENT ANTIGEN- ABILITY BINDING ABILITY NUMBER OF PANNING 2 2 NUMBEROF EXAMINED CLONES 85 86 ELISA-POSITIVE 77 75 TYPES OF ELISA-POSITIVE 7472 CLONE SEQUENCES TYPES OF Ca-DEPENDENT 13 47 BINDING CLONE SEQUENCES

(41-3) Expression and Purification of Antibodies

Clones that are determined to have Ca-dependent antigen binding abilityas a result of phage ELISA were inserted into animal cell expressionplasmids. Antibodies were expressed by the following method. Cells ofhuman fetal kidney cell-derived FreeStyle 293-F (Invitrogen) weresuspended in FreeStyle 293 Expression Medium (Invitrogen), and plated ata cell density of 1.33×10⁶ cells/ml (3 ml) into each well of a 6-wellplate. The prepared plasmids were introduced into cells by a lipofectionmethod. The cells were cultured for four days in a CO₂ incubator (37°C., 8% CO₂, 90 rpm). From the culture supernatants prepared as describedabove, antibodies were purified using the rProtein A Sepharose™ FastFlow (Amersham Biosciences) by a method known to those skilled in theart. Absorbance at 280 nm of purified antibody solutions was measuredusing a spectrophotometer. Antibody concentrations were calculated fromthe determined values using an extinction coefficient calculated by thePACE method (Protein Science (1995) 4: 2411-2423).

(41-4) Assessment of Isolated Antibodies for their Ca-Dependent BindingAbility to Human IL-6 Receptor

Antibodies 6RC1IgG_(—)010 (heavy chain SEQ ID NO: 133; light chain SEQID NO: 134), 6RC1IgG_(—)012 (heavy chain SEQ ID NO: 135; light chain SEQID NO: 136), and 6RC1IgG_(—)019 (heavy chain SEQ ID NO: 137; light chainSEQ ID NO: 138) isolated as described above were assessed for the Cadependency of their human IL-6 receptor-binding activity by analyzingthe interaction between the antibodies and human IL-6 receptor usingBiacore T100 (GE Healthcare). Tocilizumab (heavy chain SEQ ID NO: 111;light chain SEQ ID NO: 112) was used as a control antibody that does nothave Ca-dependent binding activity to human IL-6 receptor. Theinteraction was analyzed in solutions at 1.2 mM and 3 μM calcium ionconcentration, corresponding to high and low calcium ion concentrationconditions, respectively. An appropriate amount of Protein A/G(Invitrogen) was immobilized onto a Sensor chip CM5 (GE Healthcare) byan amine coupling method, and antibodies of interest were captured ontothe chip. The two types of running buffers used were: 20 mM ACES/150 mMNaCl/0.05% (w/v) Tween20/1.2 mM CaCl₂ (pH 7.4); and 20 mM ACES/150 mMNaCl/0.05% (w/v) Tween20/3 μM CaCl₂ (pH 7.4). These buffers were eachused to dilute human IL-6 receptor. All measurements were carried out at37° C.

In the interaction analysis of the antigen-antibody reaction usingantibody tocilizumab as a control antibody, and antibodies6RC1IgG_(—)010, 6RC1IgG_(—)012, and 6RC1IgG_(—)019, a diluted IL-6receptor solution and a running buffer as a blank were injected at aflow rate of 5 μl/min for three minutes to allow IL-6 receptor tointeract with antibodies tocilizumab, 6RC1IgG_(—)010, 6RC1IgG_(—)012,and 6RC1IgG_(—)019 captured onto the sensor chip. Then, 10 mMglycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30seconds to regenerate the sensor chip.

Sensorgrams at the high calcium ion concentration obtained by themeasurement using the above-described method are shown in FIG. 61.

Under the low calcium ion concentration condition, sensorgrams ofantibodies tocilizumab, 6RC1IgG_(—)010, 6RC1IgG_(—)012, and6RC1IgG_(—)019 were also obtained by the same method. Sensorgrams at thelow calcium ion concentration are shown in FIG. 62.

The result described above shows that the IL6 receptor-binding abilityof antibodies 6RC1IgG_(—)010, 6RC1IgG_(—)012, and 6RC1IgG_(—)019 wassignificantly reduced when the calcium ion concentration in the bufferwas shifted from 1.2 mM to 3

Reference Example 42 Design of pH-Dependent Binding Antibody Library

(42-1) Method for Acquiring pH-Dependent Binding Antibodies

WO2009/125825 discloses a pH-dependent antigen-binding molecule whoseproperties are changed in neutral pH and acidic pH ranges by introducinga histidine into an antigen-binding molecule. The disclosed pH-dependentantigen-binding molecule is obtained by alteration to substitute a partof the amino acid sequence of the antigen-binding molecule of interestwith a histidine. To obtain a pH-dependent antigen-binding molecule moreefficiently without preliminarily obtaining the antigen-binding moleculeof interest to be modified, one method may be obtaining anantigen-binding molecule that binds to a desired antigen from apopulation of antigen-binding molecules (referred to as His library)with a histidine introduced into the variable region (more preferably, aposition potentially involved in antigen binding). It may be possible toefficiently obtain an antigen-binding molecule having desired propertiesfrom a His library, because histidine appears more frequently inantigen-binding molecules from His library than those from conventionalantibody libraries.

(42-2) Design of a Population of Antibody Molecules (His Library) withHistidine Residue Introduced into their Variable Region to EffectivelyAcquire Antibodies that Bind to Antigen in a pH-Dependent Manner

First, positions for introducing a histidine were selected in a Hislibrary. WO 2009/125825 discloses generation of pH-dependentantigen-binding molecules by substituting amino acid residues in thesequences of IL-6 receptor antibodies, IL-6 antibodies, and IL-31receptor antibodies with a histidine. In addition, an egg white lysozymeantibody (FEBS Letter 11483, 309, 1, 85-88) and hepcidin antibody(WO2009/139822) having a pH-dependent antigen-binding ability weregenerated by substituting the amino acid sequence of the antigen-bindingmolecule with histidines. Positions where histidines were introduced inthe IL-6 receptor antibody, IL-6 antibody, IL-31 receptor antibody, eggwhite lysozyme antibody, and hepcidin antibody are shown in Table 57.Positions shown in Table 57 may be listed as candidate positions thatcan control the antigen-antibody binding. In addition, besides thepositions shown in Table 57, positions that are likely to have contactwith antigen were also considered to be suitable for introduction ofhistidines.

TABLE 57 ANTIBODY CHAIN POSITION (Kabat) IL-6 RECEPTOR H 27 31 32 35 5058 62 100B 102 ANTIBODY L 28 31 32 53 56 92 IL-6 ANTIBODY H 32 59 61 99L 53 54 90 94 IL-31 RECEPTOR H 33 ANTIBODY L EGG-WHILE LYSOZYME H 33 98ANTIBODY L 54 HEPCIDIN ANTIBODY H 52 57 99 107 L 27 89

In the His library consisting of heavy-chain and light-chain variableregions, a human antibody sequence was used for the heavy chain variableregion, and histidines were introduced into the light chain variableregion. The positions listed above and positions that may be involved inantigen binding, i.e., positions 30, 32, 50, 53, 91, 92, and 93 (Kabatnumbering, Kabat E A et al. 1991. Sequence of Proteins of ImmunologicalInterest. NIH) in the light chain were selected as positions forintroducing histidines in the His library. In addition, the hVk1sequence was selected as a template sequence of the light chain variableregion for introducing histidines. Multiple amino acids were allowed toappear in the template sequence to diversify antigen-binding moleculesthat constitute the library. Positions exposed on the surface of avariable region that is likely to interact with the antigen wereselected as those where multiple amino acids are allowed to appear.Specifically, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94, and 96of the light chain (Kabat numbering, Kabat E A et al. 1991. Sequence ofProteins of Immunological Interest. NIH) were selected as flexibleresidues.

The type and appearance frequency of amino acid residues that weresubsequently allowed to appear were determined. The appearance frequencyof amino acids in the flexible residues in the hVk1 and hVk3 sequencesregistered in the Kabat database (KABAT, E. A. ET AL.: ‘Sequences ofproteins of immunological interest’, vol. 91, 1991, NIH PUBLICATION) wasanalyzed. Based on the analysis results, the type of amino acids thatwere allowed to appear in the His library were selected from those withhigher appearance frequency at each position. At this time, amino acidswhose appearance frequency was determined to be low based on theanalysis results were also selected to avoid the bias of amino acidproperties. The appearance frequency of the selected amino acids wasdetermined in reference to the analysis results of the Kabat database.

As His libraries, His library 1 which is fixed to necessarilyincorporate a single histidine into each CDR, and His library 2 which ismore emphasized on sequence diversity than the His library 1 weredesigned by taking the amino acids and appearance frequency set asdescribed above into consideration. The detailed designs of Hislibraries 1 and 2 are shown in Tables 7 and 8 (with the positions ineach table representing the Kabat numbering). In Tables 7 and 8, Ser (S)at position 94 can be excluded if position 92 represented by the Kabatnumbering is Asn (N).

Reference Example 43 Preparation of a Phage Display Library for HumanAntibodies (His Library 1) to Obtain an Antibody that Binds to Antigenin a pH-Dependent Manner

A gene library of antibody heavy-chain variable regions was amplified byPCR using a poly A RNA prepared from human PBMC, and commercial humanpoly A RNA as a template. A gene library of antibody light-chainvariable regions designed as His library 1 as described in ReferenceExample 42 was amplified using PCR. A combination of the gene librariesof antibody heavy-chain and light-chain variable regions generated asdescribed above was inserted into a phagemid vector to construct a humanantibody phage display library which presents Fab domains consisting ofhuman antibody sequences. For the construction method, Methods Mol.Biol. (2002) 178, 87-100 was used as a reference. For the constructionof the library, the sequences of a phage display library with a trypsincleavage sequence inserted into a linker region connecting the phagemidFab to the phage pIII protein, and between the N2 and CT domains of thehelper phage pIII protein gene were used. Sequences of the antibodygenes isolated from E. coli into which the antibody gene library wasintroduced were identified, and sequence information was obtained for132 clones. The designed amino acid distribution and the amino aciddistribution of the identified sequences are shown in FIG. 63. A librarycontaining various sequences corresponding to the designed amino aciddistribution was constructed.

Reference Example 44 Isolation of Antibodies that Bind to IL-6R in apH-Dependent Manner

(44-1) Isolation of Antibody Fragments, which Bind to Antigens in apH-Dependent Manner, from the Library by Bead Panning

The first selection from the constructed His library 1 was performed byenriching only antibody fragments with antigen (IL-6R) binding ability.

Phages were produced by E. coli containing the constructed phagemids forphage display. To precipitate the phages, 2.5 M NaCl/10% PEG was addedto the E. coli culture media of phage production. The precipitated phagepopulation was diluted with TBS to prepare a phage library solution. BSAand CaCl₂ were added to the phage library solution to adjust the finalBSA concentration to 4% and the final calcium ion concentration to 1.2mM. Regarding the panning method, the present inventors referred togeneral panning methods using antigens immobilized onto magnetic beads(J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001)247 (1-2), 191-203; Biotechnol. Prog. (2002) 18(2) 212-20, Mol. Cell.Proteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to theprepared phage library solution to allow the contact of the phagelibrary solution with the antigen at room temperature for 60 minutes.BSA-blocked magnetic beads were added and allowed to bind toantigen/phage complexes at room temperature for 15 minutes. The beadswere washed three times with 1 ml of 1.2 mM CaCl₂/TBST (TBS containing1.2 mM CaCl₂ and 0.1% Tween20) and then twice with 1 ml of 1.2 mMCaCl₂/TBS (pH 7.6). Then, the beads added with 0.5 ml of 1 mg/ml trypsinwere suspended at room temperature for 15 minutes, and then immediatelyseparated using a magnetic stand to collect a phage solution. Thecollected phage solution was added to 10 ml of E. coli strain ER2738 ina logarithmic growth phase (OD600 of 0.4-0.7). The E. coli was infectedwith the phages by culturing them while gently stirring at 37° C. forone hour. The infected E. coli was plated in a 225 mm×225 mm plate.Then, the phages were collected from the culture medium of the plated E.coli to prepare a phage library solution.

To enrich the phages, the second and subsequent rounds of panning wereperformed using the antigen-binding ability or the pH-dependent bindingability as an indicator. Specifically, 40 pmol of the biotin-labeledantigen was added to the prepared phage library solution to allow thecontact of the phage library solution with the antigen at roomtemperature for 60 minutes. BSA-blocked magnetic beads were added andallowed to bind to antigen/phage complexes at room temperature for 15minutes. The beads were washed multiple times with 1 ml of 1.2 mMCaCl₂/TBST and 1.2 mM CaCl₂/TBS. Then, when the phages were enrichedusing the antigen-binding ability as an indicator, the beads added with0.5 ml of 1 mg/ml trypsin were suspended at room temperature for 15minutes, and then immediately separated using a magnetic stand tocollect a phage solution. Alternatively, when the phages were enrichedusing the pH-dependent antigen-binding ability as an indicator, thebeads added with 0.1 ml of 50 mM MES/1.2 mM CaCl₂/150 mM NaCl (pH 5.5)were suspended at room temperature, and then immediately separated usinga magnetic stand to collect a phage solution. To eliminate the abilityfrom phages displaying no Fab to infect E. coli, the pIII protein(helper phage-derived pIII protein) of phages displaying no Fab wascleaved by adding 5 μl of 100 mg/ml trypsin to the collected phagesolution. The collected phages were added to 10 ml of E. coli strainER2738 in a logarithmic growth phase (OD600 of 0.4-0.7). The E. coli wasinfected with the phages by culturing them while gently stirring at 37°C. for one hour. The infected E. coli was plated in a 225 mm×225 mmplate. Then, the phages were collected from the culture medium of theplated E. coli to collect a phage library solution. The panning usingthe antigen-binding ability or the pH-dependent binding ability as anindicator was repeated twice.

(44-2) Assessment by Phage ELISA

Phage-containing culture supernatants were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above.

To the phage-containing culture supernatants, BSA and CaCl₂ were addedat a final concentration of 4% BSA and at a final calcium ionconcentration of 1.2 mM. These phage-containing culture supernatantswere subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of PBScontaining the biotin-labeled antigen. After washing each well of theplate with PBST (PBS containing 0.1% Tween20) to remove the antigen, thewells were blocked with 250 μl of 4% BSA/TBS for one hour or more. Afterremoving 4% BSA/TBS, the prepared culture supernatants were added toeach well. The antibodies presented on the phages were allowed to bindto the antigens on each well by incubating the plate at 37° C. for onehour. Following wash with 1.2 mM CaCl₂/TBST, 1.2 mM CaCl₂/TBS (pH 7.6)or 1.2 mM CaCl₂/TBS (pH 5.5) was added to each well. The plate wasincubated at 37° C. for 30 minutes. After washing with 1.2 mMCaCl₂/TBST, HRP-coupled anti-M13 antibody (Amersham Pharmacia Biotech)diluted with TBS containing 4% BSA and 1.2 mM ionized calcium was addedto each well. The plate was incubated for one hour. After washing with1.2 mM CaCl₂/TBST, TMB single solution (ZYMED) was added to each well.The chromogenic reaction in the solution of each well was stopped byadding sulfuric acid, and then the absorbance at 450 nm was measured toassess the color development.

When enrichment was carried out using the antigen-binding ability as anindicator, phage ELISA following two rounds of panning showed that 17 of96 clones were ELISA positive in an antigen-specific manner. Thus,clones were analyzed after three rounds of panning. Meanwhile, whenenrichment was carried out using the pH-dependent antigen-bindingability as an indicator, phage ELISA following two rounds of panningshowed that 70 of 94 clones were positive in ELISA. Thus, clones wereanalyzed after two rounds of panning

The base sequences of genes amplified with specific primers wereanalyzed for the clones subjected to phage ELISA. The results of phageELISA and sequence analysis are shown in Table 58 below.

TABLE 58 LIBRARY His LIBRARY 1 His LIBRARY 1 ENRICHMENT INDEXANTIGEN-BINDING pH-DEPENDENT ANTIGEN- ABILITY BINDING ABILITY NUMBER OFPANNING 3 2 NUMBER OF EXAMINED CLONES 80 94 ELISA-POSITIVE 76 70 TYPESOF ELISA-POSITIVE 30 67 CLONE SEQUENCES TYPES OF pH-DEPENDENT 22 47BINDING CLONE SEQUENCES

By the same method, antibodies with pH-dependent antigen-binding abilitywere isolated from the naive human antibody phage display library. Whenenrichment was carried out using the antigen-binding ability as anindicator, 13 types of pH-dependent binding antibodies were isolatedfrom 88 clones tested. Meanwhile, when enrichment was carried out usingthe pH-dependent antigen-binding ability as an indicator, 27 types ofpH-dependent binding antibodies were isolated from 83 clones tested.

The result described above demonstrated that the variation of cloneswith pH-dependent antigen-binding ability isolated from the His library1 was larger as compared to the naive human antibody phage displaylibrary.

(44-3) Expression and Purification of Antibodies

Clones assumed to have pH-dependent antigen-binding ability based on theresult of phage ELISA were introduced into animal cell expressionplasmids. Antibodies were expressed using the method described below.Cells of human fetal kidney cell-derived FreeStyle 293-F line(Invitrogen) were suspended in FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)to each well of a 6-well plate. The prepared plasmids were introducedinto the cells by a lipofection method. The cells were cultured in a CO₂incubator (37° C., 8% CO₂, 90 rpm) for four days. By a method known tothose skilled in the art, antibodies were purified using rProtein ASepharose™ Fast Flow (Amersham Biosciences) from culture supernatantsobtained as described above. The absorbance of solutions of purifiedantibodies was measured at 280 nm using a spectrophotometer. Antibodyconcentrations were calculated from the measured values by using theextinction coefficient determined by PACE method (Protein Science (1995)4, 2411-2423).

(44-4) Assessment of Isolated Antibodies for their pH-Dependent BindingAbility to Human IL-6 Receptor

Antibodies 6RpH#01 (heavy chain SEQ ID NO: 139; light chain SEQ ID NO:140), 6RpH#02 (heavy chain SEQ ID NO: 141; light chain SEQ ID NO: 142),and 6RpH#03 (heavy chain SEQ ID NO: 143; light chain SEQ ID NO: 144)isolated as described in (44-3) were assessed for the pH dependency oftheir human IL-6 receptor-binding activity by analyzing the interactionbetween the antibodies and human IL-6 receptor using Biacore T100 (GEHealthcare). Tocilizumab (heavy chain SEQ ID NO: 111; light chain SEQ IDNO: 112) was used as a control antibody that does not have pH-dependentbinding activity to human IL-6 receptor. The interaction for theantigen-antibody reaction was analyzed in solutions at pH 7.4 and pH6.0, corresponding to a neutral pH and acidic pH conditions,respectively. An appropriate amount of Protein A/G (Invitrogen) wasimmobilized onto a Sensor chip CM5 (GE Healthcare) by an amine couplingmethod, and about 300 RU each of antibodies of interest were capturedonto the chip. The two types of running buffers used were: 20 mMACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl₂ (pH 7.4); and 20 mMACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl₂ (pH 6.0). Thesebuffers were each used to dilute human IL-6 receptor. All measurementswere carried out at 37° C.

In the interaction analysis of the antigen-antibody reaction usingtocilizumab as a control antibody, and antibodies 6RpH#01, 6RpH#02, and6RpH#03, a diluted IL-6 receptor solution and a running buffer as ablank were injected at a flow rate of 5 μl/min for three minutes toallow IL-6 receptor to interact with antibodies tocilizumab, 6RpH#01,6RpH#02, and 6RpH#03 captured onto the sensor chip. Then, 10 mMglycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30seconds to regenerate the sensor chip.

Sensorgrams at pH 7.4 obtained by the measurement using the methoddescribed above are shown in FIG. 64. Sensorgrams under the condition ofpH 6.0 obtained by the same method are shown in FIG. 65.

The result described above shows that the IL-6 receptor-binding abilityof antibodies 6RpH#01, 6RpH#02, and 6RpH#03 was significantly reducedwhen the buffer pH was shifted from pH 7.4 to pH 6.0.

INDUSTRIAL APPLICABILITY

The present invention has successfully obtained antigen-bindingmolecules that promote antigen elimination from blood (from serum orplasma), wherein the physiological activities of an antigen having twoor more physiological activities which are difficult to inhibit in vitrowith a single type of antigen-binding molecule can be reduced with asingle type of antigen-binding molecule in vivo. Diseases that arecaused by an antigen with multiple physiological activities have beendifficult to treat with a single type of pharmaceutical agent alone. Thepresent invention can provide effective pharmaceutical agents for suchdiseases.

1.-53. (canceled)
 54. An antigen-binding molecule comprising anantigen-binding domain and at least one receptor-binding domain, whereinat a pH in the range of 4.0 to 6.5, the receptor-binding domain hashuman neonatal Fc Receptor (FcRn)-binding activity; at a pH in the rangeof 7.0 to 9.0, the ability of the receptor-binding domain to bind to ahuman Fc receptor is greater than the ability of a native human IgG tobind to the human Fc receptor; the antigen-binding domain binds to anantigen; the ability of the antigen-binding domain to bind to theantigen varies with an ion concentration; the antigen has two or morephysiological activities; binding of the antigen-binding molecule to theantigen inhibits a first physiological activity of the antigen and doesnot inhibit a second physiological activity of the antigen; andintroduction of the antigen-binding molecule into an individual whoseplasma comprises the antigen reduces the plasma concentration of theantigen.
 55. The antigen-binding molecule of claim 54, wherein the firstphysiological activity of the antigen is mediated by the antigen'sbinding to a first target molecule; the second physiological activity ofthe antigen is mediated by the antigen's binding to a second targetmolecule that is different from the first target molecule; and theantigen-binding molecule inhibits binding of the antigen to the firsttarget molecule but not to the second target molecule.
 56. Theantigen-binding molecule of claim 54, wherein the reduction of plasmaantigen concentration is due to the promotion of antigen uptake intocells by the antigen-binding molecule.
 57. The antigen-binding moleculeof claim 54, wherein the reduction of the antigen's plasma concentrationresults in a decrease of the antigen's physiological activities in theindividual.
 58. The antigen-binding molecule of claim 54, wherein theantigen is high mobility group box 1 (HMGB1).
 59. The antigen-bindingmolecule of claim 58, wherein the antigen-binding molecule inhibits thebinding of HMGB1 to Receptor for Advanced Glycation Endproducts (RAGE).60. The antigen-binding molecule of claim 58, wherein theantigen-binding molecule inhibits the binding of HMGB1 to Toll-likereceptor 4 (TLR4).
 61. The antigen-binding molecule of claim 54, whereinthe antigen is connective tissue growth factor (CTGF).
 62. Theantigen-binding molecule of claim 54, wherein the human Fc receptor ishuman FcRn.
 63. The antigen-binding molecule of claim 62, wherein thereceptor-binding domain comprises an Fc region that differs from the Fcregion of the native IgG by amino acid substitution, insertion, ordeletion at one or more positions.
 64. The antigen-binding molecule ofclaim 63, wherein the one or more positions are selected from thefollowing positions: 234, 235, 236, 237, 238, 239, 244, 245, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 267, 270,272, 274, 279, 280, 282, 283, 284, 285, 286, 288, 289, 293, 295, 297,298, 303, 305, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318,325, 326, 327, 328, 329, 330, 332, 334, 338, 339, 340, 341, 343, 345,360, 361, 362, 375, 376, 377, 378, 380, 382, 384, 385, 386, 387, 389,390, 391, 413, 422, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436,437, 438, 440, and 442 (EU numbering).
 65. The antigen-binding moleculeof claim 63 wherein the one or more positions include at least one fromthe following group of positions (EU numbering), substituted with theindicated amino acid: the amino acid at position 234 is Arg; the aminoacid at position 235 is Gly, Lys, or Arg; the amino acid at position 236is Ala, Asp, Lys, or Arg; the amino acid at position 237 is Lys, Met, orArg; the amino acid at position 238 is Ala, Asp, Lys, Leu, or Arg; theamino acid at position 239 is Asp or Lys; the amino acid at position 244is Leu; the amino acid at position 245 is Arg; the amino acid atposition 248 is Ile or Tyr; the amino acid at position 249 is Pro; theamino acid at position 250 is Ala, Glu, Phe, Ile, Met, Gln, Ser, Val,Tip, Gly, His, Leu, Asn, or Tyr; the amino acid at position 251 is Arg,Asp, Glu, or Leu; the amino acid at position 252 is Phe, Ser, Thr, Trp,or Tyr; the amino acid at position 253 is Val; the amino acid atposition 254 is Ala, Gly, His, Ile, Gln, Ser, Val, or Thr; the aminoacid at position 255 is Ala, Asp, Phe, His, Ile, Lys, Leu, Met, Asn,Gln, Arg, Gly, Ser, Tip, Tyr, or Glu; the amino acid at position 256 isAla, Asp, Glu, Arg, Asn, Pro, Thr, Ser, or Gln; the amino acid atposition 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; theamino acid at position 258 is Asp or His; the amino acid at position 260is Ser; the amino acid at position 262 is Leu; the amino acid atposition 265 is Ala; the amino acid at position 267 is Met or Leu; theamino acid at position 270 is Lys or Phe; the amino acid at position 272is Ala, Leu, or Arg; the amino acid at position 274 is Ala; the aminoacid at position 279 is Leu, Ala, Asp, Gly, His, Met, Asn, Gln, Arg,Ser, Thr, Trp, or Tyr; the amino acid at position 280 is Ala, Gly, His,Lys, Asn, Gln, Arg, Ser, Thr, or Glu; the amino acid at position 282 isAla or Asp; the amino acid at position 283 is Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acidat position 284 is Lys; the amino acid at position 285 is Asn; the aminoacid at position 286 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met,Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or Glu; the amino acid atposition 288 is Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,Gln, Arg, Val, Trp, Tyr, or Ser; the amino acid at position 289 is His;the amino acid at position 293 is Val; the amino acid at position 295 isMet; the amino acid at position 297 is Ala; the amino acid at position298 is Gly; the amino acid at position 303 is Ala; the amino acid atposition 305 is Ala or Thr; the amino acid at position 307 is Ala, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val,Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met,Pro, Gln, or Thr; the amino acid at position 309 is Ala, Asp, Glu, Pro,His, or Arg; the amino acid at position 311 is Ala, His, Glu, Lys, Leu,Met, Ser, Val, Trp, or Ile; the amino acid at position 312 is Ala, Asp,Pro, or His; the amino acid at position 313 is Tyr or Phe; the aminoacid at position 314 is Ala, Leu, Lys, or Arg; the amino acid atposition 315 is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg,Ser, Thr, Val, Trp, Tyr, or His; the amino acid at position 316 is Ala,Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, or Asp; the amino acid at position 317 is Ala or Pro; the aminoacid at position 318 is Asn or Thr; the amino acid at position 325 isAla, Gly, Met, Leu, Ile, or Ser; the amino acid at position 326 is Asp;the amino acid at position 327 is Gly; the amino acid at position 328 isArg, Asp, Glu, or Tyr; the amino acid at position 329 is Lys or Arg; theamino acid at position 330 is Leu; the amino acid at position 332 isGlu, Phe, His, Lys, Leu, Met, Arg, Ser, Trp, or Val; the amino acid atposition 334 is Leu; the amino acid at position 338 is Ala; the aminoacid at position 339 is Asn, Thr, or Trp; the amino acid at position 340is Ala; the amino acid at position 341 is Pro; the amino acid atposition 343 is Glu, His, Lys, Gln, Arg, Thr, or Tyr; the amino acid atposition 345 is Ala; the amino acid at position 360 is His; the aminoacid at position 361 is Ala; the amino acid at position 362 is Ala; theamino acid at position 375 is Ala or Arg; the amino acid at position 376is Ala, Gly, Ile, Met, Pro, Thr, or Val; the amino acid at position 377is Lys; the amino acid at position 378 is Asp, Asn, or Val; the aminoacid at position 380 is Ala, Asn, Thr, or Ser; the amino acid atposition 382 is Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Trp, Tyr, or Val; the amino acid at position 384 is Ala; the aminoacid at position 385 is Ala, Gly, Lys, Ser, Thr, Asp, His, or Arg; theamino acid at position 386 is Arg, Asp, Ile, Met, Ser, Thr, Lys, or Pro;the amino acid at position 387 is Ala, Arg, His, Pro, Ser, Thr, or Glu;the amino acid at position 389 is Ala, Asn, Pro, or Ser; the amino acidat position 390 is Ala; the amino acid at position 391 is Ala; the aminoacid at position 413 is Ala; the amino acid at position 423 is Asn; theamino acid at position 424 is Ala or Glu; the amino acid at position 427is Asn; the amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; the amino acidat position 430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, or Tyr; the amino acid at position 431 is His orAsn; the amino acid at position 433 is Arg, Gln, His, Ile, Pro, Ser, orLys; the amino acid at position 434 is Ala, Phe, Gly, Met, His, Ser,Trp, or Tyr; the amino acid at position 435 is Lys, Arg, or Asn; theamino acid at position 436 is Ala, His, Ile, Leu, Glu, Phe, Gly, Lys,Met, Asn, Arg, Ser, Thr, Trp, or Val; the amino acid at position 437 isArg; the amino acid at position 438 is Lys, Leu, Thr, or Trp; the aminoacid at position 440 is Lys; and the amino acid at position 442 is Lys.66. The antigen-binding molecule of claim 63, wherein the native IgG isan IgG native to a nonhuman animal.
 67. The antigen-binding molecule ofclaim 63, wherein the native IgG is a native human IgG.
 68. Theantigen-binding molecule of claim 1, wherein the human Fc receptor is ahuman Fcγ receptor.
 69. The antigen-binding molecule of claim 68,wherein the receptor-binding domain comprises an Fc region that differsfrom the Fc region of the native IgG by amino acid substitution,insertion, or deletion at one or more positions.
 70. The antigen-bindingmolecule of claim 69, wherein the one or more positions are selectedfrom the following positions: 221, 222, 223, 224, 225, 227, 228, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245,246, 247, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309,311, 312, 313, 314, 315, 316, 317, 318, 320, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 341, 343,375, 376, 377, 378, 379, 380, 382, 385, 386, 387, 389, 392, 396, 421,423, 427, 428, 429, 430, 431, 433, 434, 436, 438, 440; and 442 (EUnumbering).
 71. The antigen-binding molecule of claim 69, wherein theone or more positions include at least one from the following group ofpositions (EU numbering), substituted with the indicated amino acid: theamino acid at position 221 is Lys or Tyr; the amino acid at position 222is Phe, Trp, Glu, or Tyr; the amino acid at position 223 is Phe, Trp,Glu, or Lys; the amino acid at position 224 is Phe, Trp, Glu, or Tyr;the amino acid at position 225 is Glu, Lys, or Trp; the amino acid atposition 227 is Glu, Gly, Lys, or Tyr; the amino acid at position 228 isGlu, Gly, Lys, or Tyr; the amino acid at position 230 is Ala, Glu, Gly,or Tyr; the amino acid at position 231 is Glu, Gly, Lys, Pro, or Tyr;the amino acid at position 232 is Glu, Gly, Lys, or Tyr; the amino acidat position 233 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 234 isAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr; the amino acid at position 235 is Ala, Asp, Glu,Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, orTyr; the amino acid at position 236 is Ala, Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the aminoacid at position 237 is Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid atposition 238 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 239 is Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val,Trp, or Tyr; the amino acid at position 240 is Ala, Ile, Met, or Thr;the amino acid at position 241 is Asp, Glu, Leu, Arg, Trp, or Tyr; theamino acid at position 243 is Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; theamino acid at position 244 is His; the amino acid at position 245 isAla; the amino acid at position 246 is Asp, Glu, His, or Tyr, the aminoacid at position 247 is Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, orTyr; the amino acid at position 249 is Glu, His, Gln, or Tyr; the aminoacid at position 250 is Glu or Gln; the amino acid at position 251 isPhe; the amino acid at position 254 is Phe, Met, or Tyr; the amino acidat position 255 is Glu, Leu, or Tyr; the amino acid at position 256 isAla, Met, or Pro; the amino acid at position 258 is Asp, Glu, His, Ser,or Tyr; the amino acid at position 260 is Asp, Glu, His, or Tyr; theamino acid at position 262 is Ala, Glu, Phe, Ile, or Thr; the amino acidat position 263 is Ala, Ile, Met, or Thr; the amino acid at position 264is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Trp, or Tyr; the amino acid at position 265 is Ala, Glu, Leu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, orTyr; the amino acid at position 266 is Ala, Phe, Ile, Leu, Met, or Thr;the amino acid at position 267 is Ala, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the amino acidat position 268 is Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Thr, Val, or Trp; the amino acid at position 269 is Asp,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, orTyr; the amino acid at position 270 is Glu, Phe, Gly, His, Ile, Leu,Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acid at position271 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, or Tyr; the amino acid at position 272 is Asp, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, orTyr; the amino acid at position 273 is Phe or Ile; the amino acid atposition 274 is Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,Ser, Thr, Val, Trp, or Tyr; the amino acid at position 275 is Leu orTrp; the amino acid at position 276 is Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid atposition 278 is Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, or Trp; the amino acid at position 279 is Ala; theamino acid at position 280 is Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, orTyr; the amino acid at position 281 is Asp, Lys, Pro, or Tyr; the aminoacid at position 282 is Glu, Gly, Lys, Pro, or Tyr; the amino acid atposition 283 is Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr; theamino acid at position 284 is Asp, Glu, Leu, Asn, Thr, or Tyr; the aminoacid at position 285 is Asp, Glu, Lys, Gln, Trp, or Tyr; the amino acidat position 286 is Glu, Gly, Pro, or Tyr; the amino acid at position 288is Asn, Asp, Glu, or Tyr; the amino acid at position 290 is Asp, Gly,His, Leu, Asn, Ser, Thr, Trp, or Tyr; the amino acid at position 291 isAsp, Glu, Gly, His, Ile, Gln, or Thr; the amino acid at position 292 isAla, Asp, Glu, Pro, Thr, or Tyr; the amino acid at position 293 is Phe,Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; theamino acid at position 294 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 295 isAsp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, or Tyr; the amino acid at position 296 is Ala, Asp, Glu, Gly,His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the amino acidat position 297 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro,Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid at position 298 isAla, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, orTyr; the amino acid at position 299 is Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the aminoacid at position 300 is Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp; the amino acid at position301 is Asp, Glu, His, or Tyr; the amino acid at position 302 is Ile; theamino acid at position 303 is Asp, Gly, or Tyr; the amino acid atposition 304 is Asp, His, Leu, Asn, or Thr; the amino acid at position305 is Glu, Ile, Thr, or Tyr; the amino acid at position 311 is Ala,Asp, Asn, Thr, Val or Tyr; the amino acid at position 313 is Phe; theamino acid at position 315 is Leu; the amino acid at position 317 is Gluor Gln; the amino acid at position 318 is His, Leu, Asn, Pro, Gln, Arg,Thr, Val, or Tyr; the amino acid at position 320 is Asp, Phe, Gly, His,Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr; the amino acid atposition 322 is Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp,or Tyr; the amino acid at position 323 is Ile, Leu, or Met; the aminoacid at position 324 is Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg,Thr, Val, Trp, or Tyr; the amino acid at position 325 is Ala, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, orTyr; the amino acid at position 326 is Ala, Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; the amino acidat position 327 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Thr, Val, Trp, or Tyr; the amino acid at position 328 isAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr; the amino acid at position 329 is Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;the amino acid at position 330 is Cys, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acid atposition 331 is Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val,Trp, or Tyr; the amino acid at position 332 is Ala, Asp, Glu, Phe, Gly,His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; theamino acid at position 333 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, Ser, Thr, Val, or Tyr; the amino acid at position334 is Ala, Glu, Phe, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr; the amino acid at position 335 is Asp, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; the amino acid atposition 336 is Glu, Lys, or Tyr; the amino acid at position 337 is Asp,Glu, His, or Asn; the amino acid at position 339 is Asp, Phe, Gly, Ile,Lys, Met, Asn, Gln, Arg, Ser, or Thr; the amino acid at position 376 isAla or Val; the amino acid at position 377 is Gly or Lys; the amino acidat position 378 is Asp; the amino acid at position 379 is Asn; the aminoacid at position 380 is Ala, Asn, or Ser; the amino acid at position 382is Ala or Ile; the amino acid at position 385 is Glu; the amino acid atposition 392 is Thr; the amino acid at position 396 is Asp, Glu, Phe,Ile, Lys, Leu, Met, Gln, Arg, or Tyr; the amino acid at position 421 isLys; the amino acid at position 427 is Asn; the amino acid at position428 is Phe or Leu; the amino acid at position 429 is Met; the amino acidat position 434 is Trp; the amino acid at position 436 is Ile; and theamino acid at position 440 is Gly, His, Ile, Leu, or Tyr.
 72. Theantigen-binding molecule of claim 68, wherein the human Fcγ receptor isFcγRIa, FcγRIIa, FcγRIIb, or FcγRIIIa.
 73. The antigen-binding moleculeof claim 70, wherein the one or more positions include position 238substituted with Asp and position 271 substituted with Gly (EUnumbering).
 74. The antigen-binding molecule of claim 73, wherein theone or more positions also include at least one from the following groupof positions (EU numbering): 233, 234, 237, 244, 245, 249, 250, 251,252, 254, 255, 256, 257, 258, 260, 262, 264, 265, 266, 267, 268, 269,270, 272, 279, 283, 285, 286, 288, 293, 296, 307, 308, 309, 311, 312,314, 316, 317, 318, 326, 327, 330, 331, 332, 333, 339, 341, 343, 375,376, 377, 378, 380, 382, 385, 386, 387, 389, 396, 423, 427, 428, 430,431, 433, 434, 436, 438, 440, and
 442. 75. The antigen-binding moleculeof claim 74, wherein at least one position from the following group ofpositions (EU numbering) is substituted with the indicated amino acid:the amino acid at position 233 is Asp; the amino acid at position 234 isTyr; the amino acid at position 237 is Asp; the amino acid at position264 is Ile; the amino acid at position 265 is Glu; the amino acid atposition 266 is Phe, Met, or Leu; the amino acid at position 267 is Ala,Glu, Gly, or Gln; the amino acid at position 268 is Asp or Glu; theamino acid at position 269 is Asp; the amino acid at position 272 isAsp, Phe, Ile, Met, Asn, or Gln; the amino acid at position 296 is Asp;the amino acid at position 326 is Ala or Asp; the amino acid at position327 is Gly; the amino acid at position 330 is Lys or Arg; the amino acidat position 331 is Ser; the amino acid at position 332 is Thr, the aminoacid at position 333 is Thr, Lys, or Arg; and the amino acid at position396 is Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr.
 76. Theantigen-binding molecule of claim 73, wherein the one or more positionsalso include at least one from the following group of positions (EUnumbering): 244, 245, 249, 250, 251, 252, 254, 255, 256, 257, 258, 260,262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 308, 309, 311, 312,314, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382,385, 386, 387, 389, 423, 427, 428, 430, 431, 433, 434, 436, 438, 440,and
 442. 77. The antigen-binding molecule of claim 76, wherein at leastone position from the following group of positions (EU numbering) issubstituted with the indicated amino acid: the amino acid at position244 is Leu; the amino acid at position 245 is Arg; the amino acid atposition 249 is Pro; the amino acid at position 250 is Gln or Glu; theamino acid at position 251 is Arg, Asp, Glu, or Leu; the amino acid atposition 252 is Phe, Ser, Thr, or Tyr; the amino acid at position 254 isSer or Thr; the amino acid at position 255 is Arg, Gly, Ile, or Leu; theamino acid at position 256 is Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr;the amino acid at position 257 is Ala, Ile, Met, Asn, Ser, or Val; theamino acid at position 258 is Asp; the amino acid at position 260 isSer; the amino acid at position 262 is Leu; the amino acid at position270 is Lys; the amino acid at position 272 is Leu or Arg; the amino acidat position 279 is Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr,Trp, or Tyr; the amino acid at position 283 is Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; the amino acidat position 285 is Asn; the amino acid at position 286 is Phe; the aminoacid at position 288 is Asn or Pro; the amino acid at position 293 isVal; the amino acid at position 307 is Ala, Glu, Gln, or Met; the aminoacid at position 308 is Ile, Pro, or Thr; the amino acid at position 309is Pro, the amino acid at position 311 is Ala, Glu, Ile, Lys, Leu, Met,Ser, Val, or Trp; the amino acid at position 312 is Ala, Asp, or Pro;the amino acid at position 314 is Ala or Leu; the amino acid at position316 is Lys; the amino acid at position 317 is Pro; the amino acid atposition 318 is Asn or Thr; the amino acid at position 332 is Phe, His,Lys, Leu, Met, Arg, Ser, or Trp; the amino acid at position 339 is Asn,Thr, or Trp; the amino acid at position 341 is Pro; the amino acid atposition 343 is Glu, His, Lys, Gln, Arg, Thr, or Tyr; the amino acid atposition 375 is Arg; the amino acid at position 376 is Gly, Ile, Met,Pro, Thr, or Val; the amino acid at position 377 is Lys; the amino acidat position 378 is Asp, Asn, or Val; the amino acid at position 380 isAla, Asn, Ser, or Thr; the amino acid at position 382 is Phe, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the amino acidat position 385 is Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr; the aminoacid at position 386 is Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; theamino acid at position 387 is Ala, Arg, His, Pro, Ser, or Thr; the aminoacid at position 389 is Asn, Pro, or Ser; the amino acid at position 423is Asn; the amino acid at position 427 is Asn; the amino acid atposition 428 is Leu, Met, Phe, Ser, or Thr; the amino acid at position430 is Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, or Tyr; the amino acid at position 431 is His or Asn; the aminoacid at position 433 is Arg, Gln, His, Ile, Lys, Pro, or Ser; the aminoacid at position 434 is Ala, Gly, His, Phe, Ser, Trp, or Tyr; the aminoacid at position 436 is Arg, Asn, His, Ile, Leu, Lys, Met, or Thr; theamino acid at position 438 is Lys, Leu, Thr, or Trp; the amino acid atposition 440 is Lys; and the amino acid at position 442 is Lys.
 78. Theantigen-binding molecule of claim 69, wherein the native IgG is an IgGnative to a nonhuman animal.
 79. The antigen-binding molecule of claim69, wherein the native IgG is a native human IgG.
 80. Theantigen-binding molecule of claim 54, wherein the ion concentration ishydrogen ion concentration (pH), and the antigen-binding activity islower at a first pH that is in the range of 4.0 to 6.5 than at a secondpH that is in the range of 7.0 to 9.0.
 81. The antigen-binding moleculeof claim 80, wherein the first pH is endosomal pH.
 82. Theantigen-binding molecule of claim 80, wherein the second pH is plasmapH.
 83. The antigen-binding molecule of claim 80, wherein the first pHis in the range of pH 5.5 to 6.5 and the second pH is in the range of pH7.0 to 8.0.
 84. The antigen-binding molecule of claim 80, wherein theratio of (a) the KD value for antigen binding by the antigen-bindingmolecule at the first pH, to (b) the KD value for antigen binding by theantigen-binding molecule at the second pH(KD(first pH)/KD(second pH)) is 2 or more.
 85. The antigen-bindingmolecule of claim 80, wherein the antigen-binding domain comprises atleast one His residue.
 86. The antigen-binding molecule of claim 80,wherein the antigen-binding activity is dependent not only on pH, butalso on calcium ion concentration, with antigen binding activity lowerat a first calcium ion concentration in the range of 0.1 μM to 30 μMthan at a second calcium ion concentration in the range of 100 μM to 10mM.
 87. The antigen-binding molecule of claim 54, wherein the ionconcentration is calcium ion concentration and the antigen-bindingactivity is lower at a first calcium ion concentration in the range of0.1 μM to 30 μM than at a second calcium ion concentration in the rangeof 100 to 10 mM.
 88. The antigen-binding molecule of claim 87, whereinthe first calcium ion concentration is an endosomal calcium ionconcentration.
 89. The antigen-binding molecule of claim 87, wherein thesecond calcium ion concentration is a plasma calcium ion concentration.90. The antigen-binding molecule of claim 87, wherein the first calciumion concentration is in the range of 1 μM to 5 μM and the second calciumion concentration is in the range of 0.5 mM to 2.5 mM.
 91. Theantigen-binding molecule of claim 87, wherein the ratio of (a) the KDvalue for antigen binding by the antigen-binding molecule at the firstcalcium ion concentration, to (b) the KD value for antigen binding bythe antigen-binding molecule at the second calcium ion concentration(KD(first calcium ion concentration)/KD(second calcium ionconcentration)) is 2 or more.
 92. The antigen-binding molecule of claim54, wherein the antigen-binding molecule is an antibody.
 93. Theantigen-binding molecule of claim 92, wherein the antibody is a chimericantibody, a humanized antibody, or a human antibody.
 94. Apharmaceutical composition comprising as an active ingredient theantigen-binding molecule of claim
 54. 95. The pharmaceutical compositionof claim 94, wherein the antigen is HMGB1.
 96. The pharmaceuticalcomposition of claim 94, wherein the antigen is CTGF.
 97. A method oftreatment comprising identifying a patient in need of treatment for acondition that is treatable by reducing the level of a particularantigen in blood; and administering a therapeutically effective amountof the pharmaceutical composition of claim 94 to the patient, whereinthe antigen to which the antigen-binding molecule binds is theparticular antigen.
 98. The method of claim 97, wherein the antigen isHMGB1.
 99. The method of claim 98, wherein the condition is sepsis. 100.The method of claim 97, wherein the antigen is CTGF.
 101. The method ofclaim 100, wherein the condition is fibrosis.
 102. A method forproducing the antigen-binding molecule of claim 54, the methodcomprising: (a) identifying one or more antigen-binding domains thatinhibit the first physiological activity of the antigen by binding tothe antigen, and do not inhibit the second physiological activity of theantigen; (b) of the antigen-binding domain(s) identified in (a),selecting an antigen-binding domain whose ability to bind to the antigenvaries with the concentration of the ion; (c) identifying areceptor-binding domain that (i) has human FcRn-binding activity at a pHin the range of 4.0 to 6.5, and (ii) at a pH in the range of 7.0 to 9.0,has a greater ability to bind to the human Fc receptor than does thenative human IgG; (d) preparing an antigen-binding molecule comprisingthe antigen-binding domain selected in (b) and the receptor-bindingdomain identified in (c); and (e) confirming that the antigen-bindingmolecule prepared in (d) inhibits the first physiological activity ofthe antigen by binding to the antigen and does not inhibit the secondphysiological activity of the antigen.
 103. The method of claim 102,wherein the amino acid sequence of the receptor-binding domain is notidentical to a sequence of any naturally-occurring receptor-bindingdomain.
 104. The method of claim 102, wherein (d) comprises producingrecombinant DNA encoding the antigen-binding molecule and expressing therecombinant DNA, thereby producing the antigen-binding molecule. 105.The method of claim 102, wherein the first physiological activity of theantigen is mediated by the antigen's binding to a first target molecule;the second physiological activity of the antigen is mediated by theantigen's binding to a second target molecule that is different from thefirst target molecule; and the antigen-binding molecule inhibits bindingof the antigen to the first target molecule but not to the second targetmolecule.
 106. The method of claim 102, wherein the human Fc receptor ishuman FcRn or human Fcγ receptor.